Textured surfaces for breast implants

ABSTRACT

The invention provides new devices for implantation in a patient having irregular textured surfaces, which devices show significantly improved cellular response compared to conventional smooth and textured implants, indicating that significantly improved biocompatibility would be achieved in vivo. Methods for making such new devices and surface textures are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/119,264 filed on Aug. 16, 2016, which is the U.S. national phaseentry under 35 U.S.C. § 371 of International Application No.PCT/GB2015/050438, filed on Feb. 16, 2015, each incorporated byreference in its entirety, which claims the benefit of priority to GBApplication No. 1402804.7, filed on Feb. 17, 2014.

TECHNICAL FIELD

This invention relates to biocompatible implant materials havingtextured surface topographies for reducing capsular contracture and anundesirable cellular response upon implantation into the body, withparticular application to prosthetic implants, such as silicone breastimplants. Methods for preparing such surfaces are also disclosed.

BACKGROUND

Fibrous capsule formation around soft tissue body implants remains apersistent problem for many patients following prosthesis implantation.Silicone shell breast implants are particularly troublesome withpotential development of capsular contracture, which is considered to beone of the primary reasons for device failure.

Capsular Contracture

The pathoetiology of capsular contracture formation around siliconemammary implants is both complex and enigmatic, however; it is thoughtto be an over-exaggeration of the normal foreign body reaction. Thereare a number of known risk factors for its development such asimplantation post-radiotherapy and bacterial infection around theimplant. In addition, some studies have shown an association betweencapsular contracture in the case of mammary implants with sub-glandularversus sub-muscular placement of the implant and use of smooth versustextured silicone shells.

There is a consensus that in the case of breast implants, the use oftextured silicone implants lowers the incidence of capsular contractureformation. It is thought that the extremely roughened surface of theseimplants disrupts and prevents the formation of parallel collagenbundles around the implant thus preventing thickened capsule formation,which can contract around the prosthesis and result in firmness,deformation and pain; the signs and symptoms most commonly associatedwith this pathology.

The pathoetiology of breast capsular contracture formation can beloosely viewed in two broadly distinct explanations: one school ofthought is that the initial protein adsorption and subsequent cellattachment to the mammary prosthesis in the first minutes to hours afterimplantation can dictate the extent of the subsequent foreign bodyreaction and clinical outcome through cell mediated cytokine/chemokinerelease and extracellular matrix production. Thus, the nano- andmicro-scale features on implants are important to this hypothesis as itis primarily centred on the initial cell response at a microscopic leveland involves specific cell-surface (motif-integrin) binding via adsorbedproteins from human serum. In contrast, another hypothesis, which led tothe production of textured implants, centres on the problem of parallelbundle fibres forming in the capsule tissue adjacent to the implant. Itis proposed that parallel collagen fibres within the capsule promote anincreased capsular contraction around the implant. Textured implantstherefore aim to disrupt the capsule tissue formation around theimplant, through its roughened texture such that parallel collagenfibres are unable to form and thus co-ordinated myofibroblast initiatedcontraction is inhibited. The latter hypothesis, however, neglects toconsider the initial reaction of the body to the implant and insteadfocuses on attempting to firmly integrate the implant within the breastso that the movement of the prosthesis is minimized; potentially leadingto reduced contracture. However, this approach can be viewed more asaltering the course of capsular contracture as opposed to preventing thecapsule contracture from being initiated. Furthermore, once capsularcontracture has occurred around a textured implant, surgical removalproves much more traumatic and can result in the unnecessary loss of thesurrounding breast tissue as there is excessive tissue ingrowth into themore heavily textured implant (the so called “velcro effect”).

Present methods of addressing the problems associated with adversecellular response, cell ingrowth and capsule contracture have beenapproached from two distinct directions. Researchers have for examplereported some success in avoiding adverse cellular response by usingsoft tissue matrix allografts (typically in the form of acellular dermalmatrix) to cover the implant in the implant site and thus providing ascaffold on which the patient's own cells can repopulate and vascularisethe graft, see e.g. [Davila, A. A. 2012], [Salzberg, C. A. 2012] and[Liu, D. Z. 2013]. Secondly, researchers have focussed on investigatingand refining the respective implant surfaces in order to improvecellular interactions.

The Implant Surface

The basic design and fabrication of current commercially availablebreast implants was generally conceived in the 1960's with limitedscientific consideration or evaluation in particular due to theavailable scientific know-how and technology at the time. Nonetheless,the primary concern for inventors and producers over time has beengaining approval from the relevant device regulatory authorities such asthe Food and Drug Administration (FDA) approval of silicone implantsafety in the United States. Thus, implants created to date weredesigned mostly to reduce capsular contracture formation rather thanminimisation of the foreign body reaction and as a consequence,extensive analysis of the physical, mechanical and chemical propertiesof breast implants would likely benefit from further detailedinvestigation.

A number of factors have since been considered by investigators in thisfield when designing a high performance, functional and long lastingimplant. In particular, there are a number of surface properties, whichappear to influence the response of cells to an implant both in vitroand in vivo. Included among these are the effects of surface roughness,topography, wettability and elastic modulus on cell response. Sincesilicone is transparent, highly elastic, durable, permeable to oxygen,FDA approved and extensively biologically tested, it remains the primarymaterial for breast prosthesis fabrication. The innate properties ofvulcanized silicone, in the form required for implantation (high tensilestrength and tear resistance), mean that the chemical and mechanicalsurface properties of implants are already established and resistant tochange. Thus, prior art approaches have focussed on modulating surfacetopography/roughness in attempts to alter or improve prosthesisperformance.

Surface Texture

In general, implant surfaces may have a primary surface profile made upof the surface form, which is the general shape of the material surface.For instance, the surface of a breast implant will generally adopt acurved form, perhaps with additional contours/waves which may be naturalfeatures/undulations that form as a result of the physical make-up ofthe implant. The way in which such surfaces interact with body tissue ata cellular level is however better described by reference to the surfaceroughness, which refers to the topographical texture of the primaryimplant surface on a smaller scale. Surface roughness is typicallyclassified on three distinct scales; macro roughness (1 μm and greater),micro roughness (100 nm to 1 μm) and nano roughness (1 nm to 100 nm).Each of these different roughness scales has been observed to have adistinct effect on both initial cell response (up to 24 hours) andlonger-term cell response (up to weeks and months). Of course, a surfacemay comprise one or more of these roughness levels. For instance, theprimary implant surface may contain only one of topographicalmacro-roughness (i.e. wherein surface features at a micro roughnessand/or nano roughness scale are not present), micro-roughness (i.e.wherein surface features at a macro roughness and/or nano roughnessscale are not present) and nano roughness (i.e. wherein surface featuresat a macro roughness and/or micro roughness scale are not present).Alternatively, implant surfaces may possess surface roughness on morethan one of these scales. For instance, the primary implant surface maycontain topographical macro-roughness as well as micro-roughness and/ornano-roughness. In such surfaces, the relevant features may appear asadjacent textures and/or as superimposed textures within the primarysurface profile. An example of superimposed features is wherein themacro-roughness profile further contains micro- and/or nano-roughnesstextures as secondary/tertiary roughness profiles respectively.

Surface Texture and Cellular Response

It is generally understood by researchers in the field that surfacetopography and roughness can influence cell response to a material[Schulte, V. A. 2009], [van Kooten, T. G. 1998], [Rompen, E. 2006].Research has for example shown that surface topography can influenceclinical outcomes for patients with hip replacements, dental implantsand silicone breast implants [Barnsley, G. P. 2006], [Harvey, A. G.2013], [Mendonca, G. 2008]. In particular, textured implants have beenshown to significantly reduce capsular contracture in comparison tosmooth implants [Barnsley, G. P. 2006]. However, as explained belowthere has been a great degree of difficulty in identifying key surfacefeature(s) that affect the cellular response upon implantation.

The idea of “contact guidance” first postulated by Weiss in 1934 is nowwell recognised by researchers in the field ofbiomaterial/surface-substrate interaction. It has been shown on a numberof occasions that cells are able to sense and respond to topographicalcues down to nanometre scale. The main cues for cell attachment to andspreading on a substrate (whether to native extra-cellular matrix (ECM)in vivo or a substrate in vitro) and subsequent proliferation arethrough chemokine/growth factor stimulation in addition to bothmechanical and topographical cues which cells are able to sense in theirenvironment via their filopodia.

Surface topography and degree of surface roughness (i.e. macro, micro ornano—as discussed above) can influence both cell genotype and phenotype.The roughness scale to which cells are most responsive is, however,quite complex. For instance, cell response can depend on cell type,surface substrate, surface topography and time scale. Furthermore, theoutcomes measured can also vary and include cell attachment, alignment,migration, proliferation, gene and protein expression. Therefore, thebest results for the design of a novel surface topography to initiateand encourage specific cellular response are likely to be achieved iftargeted at the particular desired cell response. As a result, thesurface features that might be important for providing desirable cellresponses in a given environment have been difficult to identify.

Therefore, new approaches are now required for the design, developmentand manufacture of textured implants in order to reduce capsularcontracture. [Harvey, A. G. 2013].

Preparation of Smooth and Textured Implants

Early approaches at manufacturing breast implants had focused on usingpolyurethane as the implant surface and had some success in minimisingcapsular contracture. However, due to health concerns, use ofpolyurethane was eventually superseded by silicone as the polymermaterial of choice due to silicone's biologically benign nature and itsFDA approval as discussed above.

Breast implants are typically formed by dipping an implant-shapedtemplate (mandrel) into liquid polymer so that it becomes uniformlycoated. Prior to curing, the implant can be subjected to a texturizingprocess such as imprinting on a mould to create a patterned texture insilicone (Mentor Siltex™ Implant). The mandrel is then placed in a hot,laminar flow cabinet to allow for the polymer to solidify around thetemplate (curing). This curing step allows for an equal amount of heatto be applied around the implant so that a homogenous surface iscreated. This process can be repeated several times to increase thethickness of the implant and the implant may then be further treatedwith a solvent if it is to be smooth (to further smooth out thesurface). Silicone breast implants are thus typically made through thissame basic process, regardless of whether they are designed to be smoothor textured.

In this regard, implant surfaces that are “smooth” do in fact usuallyexhibit an unintentional minor degree of surface roughness as a resultof fine ripples, grooves and/or other surface anomalies that are aninherent bi-product of the process by which the surfaces are prepared(for instance forming during the curing process as the liquid siliconetrickles down the mandrel under force of gravity).

Formally “textured” surfaces, however, typically comprise a heavilytextured surface topography. Such textures may be regular repeatinggeometric patterns or may be irregular in nature.

WO2009/046425 for example describes textured implant surfaces having ahighly ordered regular geometric repeating pattern (parallel bars) atthe micro- or nano-scale which are claimed to disrupt bacterial biofilmformation on the implant surface. The repeating pattern is formed byproduction of a master pattern using photolithographic techniques asapplied in semiconductor manufacture and the master pattern is then usedto contact print replicated patterns on the surface of the implant.However, whilst conventional photolithographic techniques can providesimple geometric structures such as the grooves depicted inWO2009/046425, such methods are not attractive when more complexgeometric patterns are sought (e.g. spheres, wedges) since such patternsdepend on the preparation and use of photo-masks with graded levels ofopacity through which graded levels of UV light may pass onto thephotoresist. Such photo-masks are expensive to produce and cannot bealtered once produced, meaning that each desired design/pattern requiresthe prior preparation of bespoke photo-masks.

WO95/03752 (see FIG. 4) also depicts an implant surface having a highlyordered regular geometric repeating pattern (pillars). These uniformmicro-textured surfaces may be produced by use of ion-beam thrustertechnology (see e.g. page 2 of WO95/03752). However, such uniformlypatterned implant surfaces typically lead to the orientation offibroblasts in conformity with the respective surface pattern (see e.g.paragraphs 28, 34 and FIGS. 14 and 15 of WO2009/046425). As explainedabove, however, the organised orientation of fibroblasts and,subsequently, collagen is understood to be a key stage in the promotionof fibrotic capsule contracture. Thus, while such ordering of fibroblastmight be more acceptable in external applications such as for use inwound healing, such highly ordered patterned surfaces are not thereforeideal for use in prosthetic implants, such as breast implants, which areprone to capsule formation and contracture.

A variety of irregular (i.e. non-uniform) textured implant surfaces havehowever been proposed in the literature with a range of differentcellular outcomes observed. A number of approaches to providing texturedsurfaces have however failed to reduce or prevent capsule formation andsubsequent contracture. For instance, paragraphs 86-89 and FIGS. 7 to 9of WO 2011/097499 describe a number of irregular textured surfaces,which fail to provide desirable capsule modulation. A ‘salt loss’technique is used in the production of commercially available Biocell™(Allergan, Inc.). Such surfaces are described and illustrated in moredetail in [Barr, S. 2009]. This technique results in an open-cellstructure. Implant surfaces formed by this “salt loss” technique arealso depicted in FIG. 5 of WO95/03752. Such implants are not howeverideal as introduction of foreign particles to the silicone surface maylead to detrimental effects on the silicone implant properties, forinstance if the relevant salts become encapsulated in the silicone.

An alternative technique for forming an open-cell structure involves theuse of an open cell foam or fibrous polymeric fabric to either form orimprint a pattern on the implant surface. For instance, the commerciallyavailable Siltex™ implant (Mentor), uses a mandrel with a polyurethanefoam texture that is imprinted into the silicone during curing. Similarfabric/open cell foam-based texturizing techniques are also described inUS 2011/0276134, WO 2011/097499 and US2002/0119177. If such opencell-like structures are achieved using a fabric with a uniformgeometry, then open-cell structures with small-scale irregularity butlong-distance uniformity may be achieved (see e.g. FIGS. 10 and 12 of US2011/0276134). Whilst such open cell structures are reported to achievesome success in preventing capsule formation, they also have drawbacksbecause the fine interstices and edges formed as a result of the processmay lack robustness and may break away from the implant surface underfrictional forces leading to detached silicone fragments in the body.Furthermore, the large, typically macroscopic, pores formed by suchprocesses have deep sides and pits which means that cells becomeembedded in the deep valleys of the implant and cannot migrate due tosides that are too steep for the cells to climb. Whilst this may hinderthe process of capsule formation, the cells cannot display naturalmigratory and proliferative behaviour with contact inhibition of cellswithin deep troughs of heavily textured implants. This is undesirablesince an adherent cell such as a fibroblast that is able to attach,migrate, proliferate and function on a surface with minimal stress andwithout inhibition, is likely to behave as a fibroblast would in vivowithin native ECM. Nonetheless, the deep troughs typically still allowthe eventual substantial in-growth of cells into the surface pores, butwhilst this may firmly anchor the implant in place in the body,excessive tissue in-growth may lead to difficulties later if the implanthas to be removed or replaced (for instance if capsular contractionnonetheless occurs) as a large amount of body tissue will also have tobe cut away with the implant.

WO95/03752 discloses an alternative method for producing irregularsurface topographies in silicone breast implants by adding filteredsilicone particles to the still tacky surface of the mandrel beforecuring and application of a top-coat (pages 10 to 12).

As discussed, present methods for forming irregular implant surfacestypically rely on crude and inherently unpredictable processes. Suchmethods thus provide non-reproducible surfaces, which may differsignificantly from batch to batch, leading to potentially unreliableresults. It is however desirable to be able to control the surfacefeatures with a high degree of accuracy, particularly in the case ofprosthetic implants such as breast implants, where differences in microand nano features have been shown to play an important role in cellularinteraction, biocompatibility and capsular contraction. Thus, thereremains a need for methods which provide control of irregular surfacefeatures with a higher degree of accuracy and reproducibility and/orwhich provide a higher degree of flexibility for producing differentdesigns.

As discussed above, it is therefore desirable for an implant surface topromote effective cell attachment, migration and proliferation (such aswould naturally occur within native extra-cellular matrix (ECM)) and/orto minimise the stressed cellular response (which can for example resultin cells secreting inflammatory and fibro-proliferative cytokines suchas tumour necrosis factor alpha (TNF-α) and others) with the object ofreducing implant capsular contracture formation. For cosmetic orprosthetic implants that are placed below the skin surface (typicallybelow mammary tissue in the case of breast implants) but which changethe external appearance of the body, e.g. breast implants, it is alsodesirable for the implant to be well anchored and to maintain a naturalappearance while also being easily surgically removable should somecapsular contracture arise, without having to remove a significantamount of adherent normal tissue with it.

As is evident from the above comments, there is a need to provide newand/or improved implants with surface topographies that mitigate orobviate one or more of the problems identified above. For instance,there is a desire to provide new implants which mitigate or obviatecapsular contraction, provide desirable levels of tissue anchoring andcellular in-growth, and/or which minimise the stress/inflammatoryresponse. Ideally, such surfaces should show high levels ofbiocompatibility and preferably allow the implant to retain a naturalappearance. There is also a desire for methods that can produce suchtopographical features reliably and accurately on an implant surface.

SUMMARY OF INVENTION

The inventors propose new biomimetic textured surface topographies forimplants, particularly breast implants. The inventors have found inparticular that by controlling aspects of the surface texture, forexample the surface roughness at macro, micro and/or nano scale toresemble corresponding features of the general surface topography of thebasement membrane and/or papillary dermis of human skin, desirable andindeed improved cellular response, reduced capsular contraction andappropriate cellular anchoring/in-growth could be achieved compared toconventional smooth and textured implants.

The inventors also propose novel methods for preparing textured surfacestructures on implants based on the surface topographies of biologicaltissue. Such methods allow the controlled replication of texturedsurface features and have the potential for wide applicability in thefield of texturizing implants in general.

This approach to designing implant surface structures from firstprinciples rather than by modification of currently available devices,which represents a significant departure from current trends and isexpected to have a large impact on the implant industry.

DETAILED DESCRIPTION

In an aspect of the invention is provided a synthetic implant materialcomprising textured surface, suitably an irregular textured surface,said surface characterised by having one or more of the group consistingof i) to vii):

i) a mean surface roughness Sa value of:

-   -   a) from 1 μm to 20 μm, optionally 1 to 15 μm, optionally 2 to 12        μm, optionally 3 to 9 μm, at an area scale of 1 mm×1 mm; and/or    -   b) from 0.1 μm to 5 μm, optionally 0.2 μm to 2 μm, optionally        0.2 μm to 1 μm at an area scale of 90 μm×90 μm; and/or    -   c) from 10 nm to 1 μm, optionally 30 nm to 500 nm, optionally 30        nm to 200 nm at an area scale of 10 μm×10 μm; and/or    -   d) from 2 nm to 15 nm, optionally 2 nm to 10 nm, optionally 2 nm        to 9 nm, optionally 3 nm to 9 nm, at an area scale of 1 μm×1 μm;

ii) a root mean square height Sq value of:

-   -   a) from 2 μm to 30 μm at an area scale of 1 mm×1 mm; and/or    -   b) from 0.2 μm to 5 μm, optionally 0.2 μm to 1.5 μm, optionally        0.3 μm to 1.0 μm at an area scale of 90 μm×90 μm; and/or    -   c) from 20 nm to 250 nm, optionally 30 nm to 250 nm, optionally        40 nm to 250 nm, optionally 60 nm to 200 nm at an area scale of        10 μm×10 μm; and/or    -   d) from 2 nm to 20 nm, optionally 4 nm to 12 nm, optionally 4 nm        to 11 nm, optionally 5 nm to 11 nm, optionally 6 nm to 10 nm,        optionally 6 nm to 9 nm, at an area scale of 1 μm×1 μm;

iii) a maximum peak height to trough depth Sz value of:

-   -   a) from 10 μm to 60 μm, at an area scale of 1 mm×1 mm; and/or    -   b) from 1 μm to 10 μm, optionally 1 μm to 7 μm, optionally 2 μm        to 7 μm, optionally 2 μm to 6 μm at an area scale of 90 μm×90        μm; and/or    -   c) from 0.1 μm to 2 μm, optionally 0.1 μm to 1.5 μm, optionally        0.3 μm to 1.2 μm at an area scale of 10 μm×10 μm; and/or    -   d) from 10 nm to 100 nm, optionally 30 nm to 70 nm, optionally        40 nm to 65 nm, optionally 40 nm to 60 nm, at an area scale of 1        μm×1 μm;

iv) a mean surface skewness Ssk value of:

-   -   a) from −1.0 to +1.0, optionally −0.7 to +0.7, optionally −0.5        to +0.5 at an area scale of 1 mm×1 mm; and/or    -   b) from −1.0 to +1.0, optionally −0.7 to +0.7, optionally −0.5        to +0.5 at an area scale of 90 μm×90 μm; and/or    -   c) from −0.7 to +0.7, optionally −0.5 to +0.5, optionally −0.3        to +0.3 at an area scale of 10 μm×10 μm; and/or    -   d) from −0.5 to +0.5, optionally −0.4 to +0.4, optionally −0.3        to +0.3, at an area scale of 1 μm×1 μm;

v) a mean excess kurtosis value (Sku minus 3) of:

-   -   a) from −1.0 to +1.0, optionally −0.7 to +0.7 at an area scale        of 1 mm×1 mm; and/or    -   b) from −1.0 to +1.0, optionally −0.7 to +0.7, optionally −0.5        to +0.5, optionally −0.3 to +0.3 at an area scale of 90 μm×90        μm; and/or    -   c) from −1.5 to +1.5, optionally −1.0 to +1.0, optionally −0.8        to +0.8, optionally −0.7 to +0.7, optionally −0.5 to +0.5,        optionally −0.3 to +0.3 at an area scale of 10 μm×10 μm; and/or    -   d) from −0.9 to +0.7, optionally −0.5 to +0.5, optionally −0.3        to +0.3, optionally −0.2 to +0.2, at an area scale of 1 μm×1 μm;

vi) a fractal dimension of

-   -   a) from 2.1 to 2.5, optionally 2.2 to 2.5 at an area scale of 1        mm×1 mm; and/or    -   b) from 2.1 to 2.5, optionally 2.2 to 2.4 at an area scale of 90        μm×90 μm; and/or    -   c) from 2.1 to 2.5, optionally 2.2 to 2.4 at an area scale of 10        μm×10 μm; and/or    -   d) from 2.1 to 2.5, optionally 2.1 to 2.4, optionally 2.1 to        2.35, optionally 2.2 to 2.35, at an area scale of 1 μm×1 μm;

vii) a linear (horizontal or vertical) autocorrelation length of:

-   -   a) from 20 μm to 200 μm, optionally 30 μm to 200 μm, optionally        50 μm to 200 μm, optionally 60 μm to 190 μm, at an area scale of        1 mm×1 mm; and/or    -   b) from 3 μm to 15 μm, optionally 4 μm to 15 μm, optionally 5 μm        to 13 μm, optionally 5 μm to 10 μm at an area scale of 90 μm×90        μm; and/or    -   c) from 0.5 μm to 2.5 μm, optionally 0.5 μm to 2 μm, optionally        0.6 μm to 2 μm, optionally 0.6 μm to 1.8 μm, optionally 0.7 μm        to 1.8 μm at an area scale of 10 μm×10 μm and/or    -   d) from 40 nm to 200 nm, optionally 20 nm to 180 nm, optionally        60 nm to 180 nm, optionally 50 nm to 150 nm, optionally 60 nm to        130 nm, optionally 70 nm to 130 nm at an area scale of 1 μm×1        μm.

In embodiments, the surface is characterised in having one selected fromthe following:

-   -   1. (i)    -   2. (ii)    -   3. (iii)    -   4. (iv)    -   5. (v)    -   6. (vi)    -   7. (vii)    -   8. (i) and (ii)    -   9. (i) and (iii)    -   10. (i) and (iv)    -   11. (i) and (v)    -   12. (i) and (vi)    -   13. (i) and (vii)    -   14. (ii) and (iii)    -   15. (ii) and (iv)    -   16. (ii) and (v)    -   17. (ii) and (vi)    -   18. (ii) and (vii)    -   19. (iii) and (iv)    -   20. (iii) and (v)    -   21. (iii) and (vi)    -   22. (iii) and (vii)    -   23. (iv) and (v)    -   24. (iv) and (vi)    -   25. (iv) and (vii)    -   26. (v) and (vi)    -   27. (v) and (vii)    -   28. (vi) and (vii)    -   29. (i), (ii) and (iii)    -   30. (i), (ii) and (iv)    -   31. (i), (ii) and (v)    -   32. (i), (ii) and (vi)    -   33. (i), (ii) and (vii)    -   34. (i), (iii) and (iv)    -   35. (i), (iii) and (v)    -   36. (i), (iii) and (vi)    -   37. (i), (iii) and (vii)    -   38. (i), (iv) and (v)    -   39. (i), (iv) and (vi)    -   40. (i), (iv) and (vii)    -   41. (i), (v) and (vi)    -   42. (i), (v) and (vii)    -   43. (i), (vi) and (vii)    -   44. (ii), (iii) and (iv)    -   45. (ii), (iii) and (v)    -   46. (ii), (iii) and (vi)    -   47. (ii), (iii) and (vii)    -   48. (ii), (iv) and (v)    -   49. (ii), (iv) and (vi)    -   50. (ii), (iv) and (vii)    -   51. (ii), (v) and (vi)    -   52. (ii), (v) and (vii)    -   53. (ii), (vi) and (vii)    -   54. (iii), (iv) and (v)    -   55. (iii), (iv) and (vi)    -   56. (iii), (iv) and (vii)    -   57. (iii), (v) and (vi)    -   58. (iii), (v) and (vii)    -   59. (iii), (vi) and (vii)    -   60. (iv), (v) and (vi)    -   61. (iv), (v) and (vii)    -   62. (iv), (vi) and (vii)    -   63. (v), (vi) and (vii)    -   64. (i), (ii), (iii) and (iv)    -   65. (i), (ii), (iii) and (v)    -   66. (i), (ii), (iii) and (vi)    -   67. (i), (ii), (iii) and (vii)    -   68. (i), (ii), (iv) and (v)    -   69. (i), (ii), (iv) and (vi)    -   70. (i), (ii), (iv) and (vii)    -   71. (i), (ii), (v) and (vi)    -   72. (i), (ii), (v) and (vii)    -   73. (i), (ii), (vi) and (vii)    -   74. (i), (iii), (iv) and (v)    -   75. (i), (iii), (iv) and (vi)    -   76. (i), (iii), (iv) and (vii)    -   77. (i), (iii), (v) and (vi)    -   78. (i), (iii), (v) and (vii)    -   79. (i), (iii), (vi) and (vii)    -   80. (i), (iv), (v) and (vi)    -   81. (i), (iv), (v) and (vii)    -   82. (i), (iv), (vi) and (vii)    -   83. (i), (v), (vi) and (vii)    -   84. (ii), (iii), (iv) and (v)    -   85. (ii), (iii), (iv) and (vi)    -   86. (ii), (iii), (iv) and (vii)    -   87. (ii), (iii), (v) and (vi)    -   88. (ii), (iii), (v) and (vii)    -   89. (ii), (iii), (vi) and (vii)    -   90. (ii), (iv), (v) and (vi)    -   91. (ii), (iv), (v) and (vii)    -   92. (ii), (iv), (vi) and (vii)    -   93. (ii), (v), (vi) and (vii)    -   94. (iii), (iv), (v) and (vi)    -   95. (iii), (iv), (v) and (vii)    -   96. (iii), (iv), (vi) and (vii)    -   97. (iii), (v), (vi) and (vii)    -   98. (iv), (v), (vi) and (vii)    -   99. (i), (ii), (iii), (iv) and (v)    -   100. (i), (ii), (iii), (iv) and (vi)    -   101. (i), (ii), (iii), (iv) and (vii)    -   102. (i), (ii), (iii), (v) and (vi)    -   103. (i), (ii), (iii), (v) and (vii)    -   104. (i), (ii), (iii), (vi) and (vii)    -   105. (i), (iii), (iv), (v) and (vi)    -   106. (i), (iii), (iv), (v) and (vii)    -   107. (i), (iii), (iv), (vi) and (vii)    -   108. (i), (iii), (v), (vi) and (vii)    -   109. (i), (iv), (v), (vi) and (vii)    -   110. (ii), (iii), (iv), (v) and (vi)    -   111. (ii), (iii), (iv), (v) and (vii)    -   112. (ii), (iii), (iv), (vi) and (vii)    -   113. (ii), (iii), (v), (vi) and (vii)    -   114. (ii), (iv), (v), (vi) and (vii)    -   115. (iii), (iv), (v), (vi) and (vii)    -   116. (i), (ii), (iii), (iv), (v) and (vi)    -   117. (i), (ii), (iii), (iv), (v) and (vii)    -   118. (i), (ii), (iii), (iv), (vi) and (vii)    -   119. (i), (ii), (iii), (v), (vi) and (vii)    -   120. (i), (ii), (iv), (v), (vi) and (vii)    -   121. (i), (iii), (iv), (v), (vi) and (vii)    -   122. (ii), (iii), (iv), (v), (vi) and (vii)    -   123. (i), (ii), (iii), (iv), (v), (vi) and (vii)

In embodiments, the surface is characterised in having one selected fromthe following:

-   -   (A) any one of (i) to (vii)    -   (B) any two of (i) to (vii)    -   (C) any three of (i) to (vii)    -   (D) any four of (i) to (vii)    -   (E) any five of (i) to (vii)    -   (F) any six of (i) to (vii)    -   (G) all of (i) to (vii)

Thus, in embodiments, the surface is characterised in having oneselected from the following:

-   -   (A) any one of 1. to 7. above    -   (B) any one of 8. to 28. above    -   (C) any one of 29. to 63. above    -   (D) any one of 64. to 98. above    -   (E) any one of 99. to 115. above    -   (F) any one of 116. to 122. above    -   (G) 123. above

In embodiments, the surface has (i) a mean surface roughness Sa asdescribed above and is characterised in having surface propertiesselected from the following:

1, 8 to 13, 29 to 43, 64 to 83, 99 to 109, 116 to 121, and 123.

In embodiments, the surface has (ii) a mean surface roughness Sq asdescribed above and is characterised in having surface propertiesselected from the following:

-   -   4, 10, 15, 19, 30, 34, 38 to 40, 44, 48 to 50, 54 to 56, 60 to        62, 64, 68 to 70, 74 to 76, 80 to 82, 84 to 86, 90 to 92, 94 to        96, 99 to 101, 105 to 107, 109 to 112, 114 to 118, and 120 to        123.

In embodiments, the surface has (iii) a maximum peak height to troughdepth Sz as described above and is characterised in having surfaceproperties selected from the following:

-   -   5, 11, 16, 20, 23, 31, 35, 38, 41 to 42, 45, 48, 51 to 52, 54,        57 to 58, 60 to 61, 65, 68, 71 to 72, 74, 77 to 78, 80 to 81, 83        to 84, 87 to 88, 90 to 91, 93 to 95, 97 to 99, 102 to 103, 105        to 106, 108 to 111, 113 to 117, and 119 to 123.

In embodiments, the surface has (iv) a mean surface skewness Ssk asdescribed above and is characterised in having surface propertiesselected from the following:

-   -   2, 14 to 18, 29 to 33, 44 to 53, 64 to 73, 84 to 93, 99 to 104,        110 to 114, 116 to 120 and 123.

In embodiments, the surface has (v) a mean excess kurtosis value (Skuminus 3) as described above and is characterised in having surfaceproperties selected from the following:

-   -   2, 14 to 18, 29 to 33, 44 to 53, 64 to 73, 84 to 93, 99 to 104,        110 to 114, 116 to 120, and 123.

In embodiments, the surface has (vi) a fractal dimension as describedabove and is characterised in having surface properties selected fromthe following:

-   -   6, 12, 17, 21, 24, 26, 28, 32, 36, 39, 41, 43, 46, 49, 51, 53,        55, 57, 59 to 60, 62 to 63, 66, 69, 71, 73, 75, 77, 79 to 80, 82        to 83, 85, 87, 89 to 90, 92 to 94, 96 to 98, 100, 102, 104 to        105, 107 to 110, 112 to 116, 118 to 123.

In embodiments, the surface has (vii) a linear (horizontal or vertical)autocorrelation length as described above and is characterised in havingsurface properties selected from the following:

-   -   7, 13, 18, 22, 25, 27 to 28, 33, 37, 40, 42 to 43, 47, 50, 52 to        53, 56, 58 to 59, 61 to 63, 67, 70, 72 to 73, 76, 78 to 79, 81        to 83, 86, 88 to 89, 91 to 93, 95 to 98, 101, 103 to 104, 106 to        109, 111 to 115, 117 to 123.

From within each of (i) to (vii) described above, the textured surfacemay have one or more of a), b), c) and d). Specifically, the texturedsurface may have, for each of (i) to (vii) any one selected from thefollowing:

-   -   a)    -   b)    -   c)    -   d)    -   a) and b)    -   a) and c)    -   a) and d)    -   a), b) and c)    -   a), b) and d)    -   a), c) and d)    -   a), b), c) and d)    -   b) and c)    -   b) and d)    -   b), c) and d)    -   c) and d)

In an aspect of the invention is provided a synthetic implant materialcomprising a textured surface, said surface having (i). In particular,the surface is characterised in having a feature or features selectedfrom the following (i) a); (i) b); (i) c); (i) d); (i) a) and b); (i) a)and c); (i) a) and d); (i) a), b) and c); (i) a), b) and d); (i) a), c)and d); (i) a), b), c) and d); (i) b) and c); (i) b) and d); and (i) b),c) and d).

In an aspect of the invention is provided a synthetic implant materialcomprising a textured surface, said surface having (ii). In particular,the surface is characterised in having a feature or features selectedfrom the following (ii) a); (ii) b); (ii) c); (ii) d); (ii) a) and b);(ii) a) and c); (ii) a) and d); (ii) a), b) and c); (ii) a), b) and d);(ii) a), c) and d); (ii) a), b), c) and d); (ii) b) and c); (ii) b) andd); and (ii) b), c) and d).

In an aspect of the invention is provided a synthetic implant materialcomprising a textured surface, said surface having (iii). In particular,the surface is characterised in having a feature or features selectedfrom the following (iii) a); (iii) b); (iii) c); (iii) d); (iii) a) andb); (iii) a) and c); (iii) a) and d); (iii) a), b) and c); (iii) a), b)and d); (iii) a), c) and d); (iii) a), b), c) and d); (iii) b) and c);(iii) b) and d); and (iii) b), c) and d).

In an aspect of the invention is provided a synthetic implant materialcomprising a textured surface, said surface having (iv). In particular,the surface is characterised in having a feature or features selectedfrom the following (iv) a); (iv) b); (iv) c); (iv) d); (iv) a) and b);(iv) a) and c); (iv) a) and d); (iv) a), b) and c); (iv) a), b) and d);(iv) a), c) and d); (iv) a), b), c) and d); (iv) b) and c); (iv) b) andd); and (iv) b), c) and d).

In an aspect of the invention is provided a synthetic implant materialcomprising a textured surface, said surface having (v). In particular,the surface is characterised in having a feature or features selectedfrom the following (v) a); (v) b); (v) c); (v) d); (v) a) and b); (v) a)and c); (v) a) and d); (v) a), b) and c); (v) a), b) and d); (v) a), c)and d); (v) a), b), c) and d); (v) b) and c); (v) b) and d); and (v) b),c) and d).

In an aspect of the invention is provided a synthetic implant materialcomprising a textured surface, said surface having (vi). In particular,the surface is characterised in having a feature or features selectedfrom the following (vi) a); (vi) b); (vi) c); (vi) d); (vi) a) and b);(vi) a) and c); (vi) a) and d); (vi) a), b) and c); (vi) a), b) and d);(vi) a), c) and d); (vi) a), b), c) and d); (vi) b) and c);

(vi) b) and d); and (vi) b), c) and d).

In an aspect of the invention is provided a synthetic implant materialcomprising a textured surface, said surface having (vii). In particular,the surface is characterised in having a feature or features selectedfrom the following (vii) a); (vii) b); (vii) c); (vii) d); (vii) a) andb); (vii) a) and c); (vii) a) and d); (vii) a), b) and c); (vii) a), b)and d); (vii) a), c) and d); (vii) a), b), c) and d); (vii) b) and c);(vii) b) and d); and (vii) b), c) and d).

Each of the areal (S) surface texture parameters (i) to (vii) discussedherein is described in ISO 25178-2: 2012(E). The measurement andcalculation methodology used to arrive at the values for the parametersis discussed below.

As noted above, suitably the textured surface is an irregular texturedsurface.

Suitably, the implant material is a synthetic implant material (e.g. anartificial implant material), and in preferred embodiments is abiomimetic material.

Area Scales/Resolution in Surfaces of the Invention

The implant materials described herein may thus include macro-surfaceroughness features, such as described by a) above, and/or microroughness features, such as described by b) above, and/or nano surfacefeatures, such as described by c) and/or d) above.

Thus, in embodiments of any of the aspects herein, for each of thescale-dependent features a) to d), said surface may have a), b), c) ord), for instance a). Alternatively, the surface may have b). The surfacemay on the other hand have c). Alternatively, the surface may have d).In other embodiments, the surface may have more than one, e.g. 2, 3 or 4of the roughness features a) to d). In embodiments, the surface hasa)+b), such as where the surface does not include c) or d). In otherembodiments, the surface has a)+c), such as where the surface does notinclude b) or d). In other embodiments the surface has a)+d), such aswhere the surface does not include b) or c). In further embodiments, thesurface has b)+c), such as where the surface do not include a) or d). Inother embodiments the surface has b)+d), such as where the surface doesnot include a) or c). In other embodiments, the surface has a)+b)+c),but not d). In other embodiments, the surface has a)+c)+d), but not b).In other embodiments, the surface has a) +b)+d), but not c). In otherembodiments, the surface has b)+c)+d), but not a). In other embodiments,the surface has a)+b)+c)+d).

The implant material of the invention may have a surface comprising oneor more of these roughness scales at discrete, e.g. adjacent, parts ofthe implant. Typically however, where more than one, for instance two,three or four of the respective area scales are provided on the implant,they are superimposed to provide a complex surface with a primarysurface topography corresponding to the larger roughness value (e.g. a)or b)), a secondary surface topography superimposed onto the primarysurface topography and optionally a tertiary surface topographysuperimposed onto the secondary surface topography. Where the primarytopography is formed by surface features at area scale a), a secondarytopography may be provided according to area scale b) and/or c).Similarly, if the primary topography is formed by surface features atarea scale b), a secondary topography may be provided according to areascale c). A tertiary surface topography may be provided according toarea scale d).

Implant material according to the invention may optionally also comprisesurface waviness, gradients or contours at a comparatively large-scaleperspective on which the surface roughness features discussed herein aresuperimposed.

As explained above, surface roughness amongst other things is known tohave an effect on cellular response upon implantation in the body.Surface roughness in the general micron-scale is thought to play a keyrole in disrupting the capsular formation and contraction by disruptingthe alignment and organisation of fibroblasts. Moreover, the implantsurfaces of the present invention may show one or more of improvedcellular attachment, proliferation and survival and altered genotypicresponse. Data comparing implants of the present invention with“conventional” comparative smooth and textured implant surfaces (seecellular response data in examples) demonstrates that these valuabletechnical effects have been achieved.

In particular, the inventors have found that the novel textured surfacesexhibit diminished inflammatory genotype and cytokine profile for cellson the surface.

Without wishing to be bound by theory, the inventors propose that theimproved results are a direct result of the novel surface roughnessfeatures of the invention. The present surfaces are on averagecomparatively rougher than commercial smooth implants, but significantlyless rough than comparative rough (commercially available textured)implants. The inventors submit that the improved cellular proliferationexhibited by these novel implant surfaces is a direct result of this“tailored” surface roughness. In particular, whilst organised capsuleformation and contracture may be prevented by the novel surfaces, thevertical height and sloped contours of the surfaces allow usual contactguidance and cellular mobility processes to continue, providing alargely natural environment for the cells and reducing the cell stressresponse. This was unexpected, especially given the abundance oftextured implants having substantially greater roughness in comparison,especially open cell foams which often typically have surface roughnessfeatures at the 100 micron-1000 micron scale and which large scaleroughness features were conventionally thought to be crucial to theirfunction (see e.g. column 5, paragraph 63 of US2011/0276134).

Even more advantageously, the controlled texture roughness of theimplant surfaces of the present claims not only means that reducedcapsular contraction, enhanced cellular proliferation and immuneresponse can be achieved, but the reduced level of roughness compared toconventional textured implants means that extensive cellular in-growthmay not occur, meaning that the implant can be removed more easily laterbecause very little of the patient's own tissue would also need to besurgically excised, minimising unnecessary tissue loss and thepost-operative trauma.

The data obtained for the implant surfaces of the present claims alsoindicates the importance of nano-scale features to the cellularresponse. In particular, material of the invention prepared by a lowerresolution fabrication method of the present invention (and so lackingin roughness features on the smaller nano-scale) showed similarpotential compared to surfaces of the present invention having finernano features prepared by a higher resolution casting method of thepresent invention), but surfaces having nano-features showed better cellproliferation and survival data. The inventors propose that thisreflects the ability of the cells to recognise nano-scale features andin particular for the biomimetic nano-scale roughness features toproduce a more natural environment and less stressed response.

Surface Roughness

Mean Surface Roughness (Sa)

In embodiments of any of the aspects herein, the implant material at anarea scale of 1 mm×1 mm comprises a mean surface roughness Sa value offrom 1 μm to 20 μm, suitably μm to 15 μm, suitably 2 μm to 12 μm,suitably 3 μm to 9 μm, suitably 4 μm to 8 μm, for instance 5 to 7 μm. Inembodiments, the surface roughness Sa value is 15 μm or less or less atthis area scale, more typically less than 12 μm, suitably less than 10μm, suitably less than 9 μm, such as less than 8 μm, preferably around 7μm. In embodiments, the surface roughness Sa value is 1 μm or more atthis area scale, suitably 2 μm or more, suitably 3 μm or more.

In embodiments of any of the aspects herein, the implant material at anarea scale of 90 μm×90 μm comprises a mean surface roughness Sa value offrom 0.1 μm to 5 μm, suitably 0.2 μm to 2 μm, suitably 0.2 μm to 1 μm,suitably 0.1 μm to 0.9 μm, for instance 0.2 μm to 0.8 μm, such as 0.3 μmto 0.7 μm, such as 0.4 to 0.6 μm. In embodiments, the surface roughnessSa value is 5 μm or less at this area scale, more typically less than 2μm, suitably less than 1 μm, suitably less than 0.6 μm, such as about0.5 μm. In embodiments, the surface roughness Sa value is 0.1 μm or moreat this area scale, suitably 0.2 μm or more, suitably 0.3 μm or more,suitably 0.4 μm or more.

In embodiments of any of the aspects herein, the implant material at anarea scale of 10 μm×10 μm comprises a mean surface roughness Sa value offrom 10 nm to 1000 nm, suitably 10 nm to 500 nm, suitably 30 nm to 500nm, suitably 30 nm to 200 nm, for instance 30 nm to 140 nm, such as 50nm to 100 nm. In embodiments, the surface roughness Sa value is 300 nmor less at this area scale, suitably 200 nm or less, suitably 150 nm orless, e.g. about 65 nm to 100 nm. In embodiments, the surface roughnessSa value is 10 nm or more at this area scale, suitably 30 nm or more,suitably 40 nm or more, suitably 50 nm or more, suitably 60 nm or more,e.g. about 65 nm to 100 nm.

In embodiments of any of the aspects herein, the implant material at anarea scale of 1 μm×1 μm comprises a mean surface roughness Sa value offrom 2 nm to 15 nm, suitably 2 nm to 10 nm, suitably 2 nm to 9 nm,suitably 3 nm to 9 nm, for instance 4 nm to 7 nm, such as 5 nm to 7 nm.In embodiments, the surface roughness Sa value is 10 nm or less at thisarea scale, suitably 9 nm or less, suitably 8 nm or less, suitably 7 nmor less, e.g. about 4 nm to 7 nm. In embodiments, the surface roughnessSa value is 2 nm or more at this area scale, suitably 3 nm or more,suitably 4 nm or more, e.g. about 4 nm to 7 nm.

For example, in embodiments, the implant material comprises a meansurface roughness Sa value of:

-   -   a) from 4 μm to 8 μm at an area scale of 1 mm×1 mm; and/or    -   b) from 0.1 μm to 0.9 μm at an area scale of 90 μm×90 μm; and/or    -   c) from 10 nm to 200 nm at an area scale of 10 μm×10 μm; and/or    -   d) from 2 nm to 8 nm at an area scale of 1 μm×1 μm.

In embodiments, the implant material according to any of the aspectsherein has a mean surface roughness Sa value of from 2 nm to 15 nm at anarea scale of 1 μm×1 μm, for instance 3 nm to 10 nm, such as from 4 nmto 9 nm, e.g. around 6 nm. In typical embodiments, the surface roughnessSa value is 20 nm or less at this area scale, more typically less than15 nm, such as less than 10 nm.

In preferred embodiment, the implant surface Sa value is substantiallyas disclosed in any one or more of FIGS. 16A to 18D, and 26A to 41.

Embodiments have, in addition to the Sa value(s) described above, one ormore of the Sq value(s) described herein and/or one or more of the Szvalue(s) described herein and/or one or more of the Ssk value(s)described herein and/or one or more of the kurtosis (Sku minus 3)value(s) described herein and/or one or more of the fractal dimension(FD) value(s) described herein and/or one or more of the surface linearauto correlation function (ACF) value(s) described herein.

Root Mean Square Height (Sq)

In embodiments of any of the aspects herein, the implant material at anarea scale of 1 mm×1 mm comprises a root mean square height Sq value offrom 2 μm to 30 μm, suitably 4 to 15 μm, such as from 5 to 10 μm. Inembodiments, the Sq value is 30 μm or less, suitably 20 μm or less,suitably 15 μm or less at this area scale, more typically less than 12μm, suitably 10 μm or less, suitably 9 μm or less, suitably 8 μm orless, e.g. 4 μm to 8 μm. In embodiments, the Sq value is 2 μm or more atthis area scale, suitably 3 μm or more, suitably 4 μm or more, suitably5 μm or more, e.g. 5 μm to 8 μm.

In embodiments of any of the aspects herein, the implant material at anarea scale of 90 μm×90 μm comprises a root mean square height Sq valueof from 0.2 μm to 5 μm, suitably 0.2 μm to 1.5 μm, suitably 0.3 μm to 1μm, suitably 0.4 μm to 0.9 μm, suitably 0.5 to 0.8 μm, e.g. about 0.5 to0.7 μm. In embodiments, the Sq value is 5 μm or less at this area scale,suitably 3 μm or less, suitably 2 μm or less, suitably 1.5 μm or less,suitably 1 μm or less, suitably 0.8 μm or less, suitably 0.7. Inembodiments, the Sq value is 0.2 μm or more at this area scale, suitably0.3 μm or more, suitably 0.4 μm or more, suitably 0.5 μm or more.

In embodiments of any of the aspects herein, the implant material at anarea scale of 10 μm×10 μm comprises a root mean square height Sq valueof from 20 nm to 250 nm, suitably 30 nm to 250 nm, suitably 40 nm to 250nm, suitably 50 nm to 200 nm, suitably 60 nm to 200 nm, e.g. 80 nm to140 nm. In embodiments, the Sq value is 250 nm or less at this areascale, suitably 200 nm or less, suitably 180 nm or less, suitably 160 nmor less, suitably 140 nm or less, suitably 130 nm or less. Inembodiments, the Sq value is 20 nm or more at this area scale, suitably30 nm or more, suitably 40 nm or more, suitably 50 nm or more, suitably60 nm or more, suitably 70 nm or more, suitably 80 nm or more.

In embodiments of any of the aspects herein, the implant material at anarea scale of 1 μm×1 μm comprises a root mean square height Sq value offrom 2 nm to 20 nm, suitably 2 nm to 12 nm, suitably 3 nm to 10 nm,suitably 4 nm to 10 nm, suitably 5 nm to 10 nm, suitably 6 nm to 10 nm,e.g. 6 nm to 9 nm. In embodiments, the Sq value is 12 nm or less at thisarea scale, suitably 11 nm or less, suitably 10 nm or less, suitably 9nm or less, suitably 8 nm or less. In embodiments, the Sq value is 2 nmor more at this area scale, suitably 3 nm or more, suitably 4 nm ormore, suitably 5 nm or more, suitably 6 nm or more, suitably 7 nm ormore.

For example, in embodiments, the implant material comprises a root meansquare height Sq value of:

-   -   a) from 2 μm to 20 μm at an area scale of 1 mm×1 mm;    -   b) from 0.2 μm to 1.5 μm at an area scale of 90 μm×90 μm; and/or    -   c) from 20 nm to 250 nm at an area scale of 10 μm×10 μm; and/or    -   d) from 4 nm to 12 nm at an area scale of 1 μm×1 μm.

In preferred embodiments, the implant surface Sq value is substantiallyas disclosed in any one or more of FIGS. 16A to 18D, and 26A to 41.

Embodiments have, in addition to the Sq value(s) described above, one ormore of the Sa value(s) described herein and/or one or more of the Szvalue(s) described herein and/or one or more of the Ssk value(s)described herein and/or one or more of the kurtosis (Sku minus 3)value(s) described herein and/or one or more of the fractal dimension(FD) value(s) described herein and/or one or more of the surface linear(horizontal or vertical) auto correlation function (ACF) value(s)described herein.

Maximum Peak Height to Trough Depth (Sz)

In embodiments of any of the aspects herein, the implant material at anarea scale of 1 mm×1 mm comprises a mean Sz value of from 10 μm to 80μm, suitably 10 μm to 60 μm, suitably 20 μm to 60 μm, suitably 30 μm to60 μm, suitably 30 to 50 μm, e.g. 35 μm to 45 μm. In embodiments, the Szvalue is 80 μm or less at this area scale, suitably 60 μm or less,suitably 55 μm or less, suitably 50 μm or less, suitably 48 μm or less,suitably 45 μm or less. In embodiments, the Sz value is 5 μm or more atthis area scale, suitably 10 μm or more, suitably 15 μm or more,suitably 20 μm or more, suitably 30 μm or more, suitably 35 μm or more,suitably 40 μm or more.

In embodiments of any of the aspects herein, the implant material at anarea scale of 90 μm×90 μm comprises a mean Sz value of from 1 μm to 10μm, suitably 1 μm to 8 μm, suitably 2 μm to 8 μm, suitably 2 μm to 7 μm,suitably 3 μm to 6 μm, e.g. about 3 μm to 5 μm. In embodiments, the Szvalue is 10 μm or less at this area scale, suitably 8 μm or less,suitably 7 μm or less, suitably 6 μm or less, suitably 5 μm or less. Inembodiments, the Sz value is 1 μm or more at this area scale, suitably 2μm or more, suitably 3 μm or more.

In embodiments of any of the aspects herein, the implant material at anarea scale of 10 μm×10 μm comprises a mean Sz value of from 0.1 μm to 2μm, suitably 0.1 μm to 1.5 μm, suitably 0.3 μm to 1.2 μm, suitably 0.4μm to 0.8 μm, e.g. about 0.5 μm to 0.8 μm. In embodiments, the Sz valueis 2 μm or less at this area scale, suitably 1.5 μm or less, suitably1.2 μm or less, suitably 1 μm or less, suitably 0.9 μm or less, suitably0.8 μm or less.

In embodiments of any of the aspects herein, the implant material at anarea scale of 1 μm×1 μm comprises a mean Sz value of from 10 nm to 100nm, suitably 30 nm to 70 nm, suitably 40 nm to 65 nm, suitably 40 nm to60 nm, suitably 40 nm to 55 nm, e.g. about 45 nm to 55 nm. Inembodiments, the Sz value is 70 nm or less at this area scale, suitably65 nm or less, suitably 60 nm or less, suitably 55 nm or less. Inembodiments, the Sz value is 30 nm or more at this area scale, suitably35 nm or more, suitably 40 nm or more, suitably 45 nm or more.

For example, in embodiments, the implant material comprises an Sz valueof:

-   -   a) from 10 μm to 60 μm, optionally 30 μm to 50 μm at an area        scale of 1 mm×1 mm;    -   b) from 2 μm to 8 μm at an area scale of 90 μm×90 μm; and/or    -   c) from 0.1 μm to 1.5 μm at an area scale of 10 μm×10 μm; and/or    -   d) from 30 nm to 70 nm at an area scale of 1 μm×1 μm.

In preferred embodiments, the implant surface Sz value is substantiallyas disclosed in any one or more of FIGS. 16A to 18D, and 26A to 41.

Embodiments have, in addition to the Sz value(s) described above, one ormore of the Sa value(s) described herein and/or one or more of the Sqvalue(s) described herein and/or one or more of the Ssk value(s)described herein and/or one or more of the kurtosis (Sku minus 3)value(s) described herein and/or one or more of the fractal dimension(FD) value(s) described herein and/or one or more of the surface linear(horizontal or vertical) auto correlation function (ACF) value(s)described herein.

Skewness (Ssk)

In embodiments of any of the aspects herein the ratio of the averagepeak height to average trough depth at the respective area scale is from2:3 to 3:2, optionally from 4.5:5.5 to 5.5:4.5, optionally wherein theaverage peak height and average trough height are substantially equal.

In embodiments, the distribution of surface peak relative to troughheights is substantially symmetrical about the mean height value at therespective scales, optionally wherein the surface heights aresubstantially normally distributed about the mean height value at therespective scales.

In embodiments of any of the aspects herein, the implant material at anarea scale of 1 mm×1 mm comprises a mean Ssk value of from −1.0 to 1.0,suitably −0.8 to 0.8, suitably −0.7 to 0.7, suitably −0.5 to 0.5,suitably −0.4 to 0.4, suitably −0.3 to 0.3, suitably −0.2 to 0.2,suitably −0.15 to 0.15, e.g. about zero. In embodiments, the Ssk valueis 1.0 or less at this area scale, suitably 0.8 or less, suitably 0.6 orless, suitably 0.5 or less, suitably 0.4 or less, suitably 0.3 or less,suitably 0.25 or less, suitably 0.2 or less, suitably 0.15 or less. Inembodiments, the Ssk value is −0.5 or more at this area scale, suitably−0.4 or more, suitably −0.3 or more, suitably −0.2 or more, suitably−0.1 or more, suitably 0 or more, suitably 0.05 or more.

In embodiments of any of the aspects described herein, the implantmaterial at an area scale of 90 μm×90 μm comprises a mean Ssk value offrom −1.0 to 1.0, suitably −0.9 to 0.9, suitably −0.8 to 0.8, suitably−0.6 to 0.6, suitably −0.4 to 0.4, suitably −0.3 to 0.3, suitably −0.2to 0.2, suitably −0.15 to 0.15, e.g. around zero. In embodiments, theSsk value is 1.0 or less at this area scale, suitably 0.8 or less,suitably 0.6 or less, suitably 0.4 or less, suitably 0.3 or less,suitably 0.25 or less, suitably 0.2 or less, suitably 0.18 or less,suitably 0.15 or less. In embodiments, the Ssk value is −1.0 or more atthis area scale, suitably −0.8 or more, suitably −0.6 or more, suitably−0.4 or more, suitably −0.2 or more, suitably −0.1 or more, suitably−0.08 or more.

In embodiments of any aspects herein, the implant material at an areascale of 10 μm×10 μm comprises a mean Ssk value of from −0.8 to 0.8,suitably −0.7 to 0.7, suitably −0.6 to 0.6, suitably −0.5 to 0.5,suitably −0.4 to 0.4, suitably −0.3 to 0.3, suitably −0.2 to 0.2, e.g.around zero. In embodiments, the Ssk value is 0.8 or less at this areascale, suitably 0.7 or less, suitably 0.6 or less, suitably 0.5 or less,suitably 0.4 or less, suitably 0.3 or less, suitably 0.2 or less,suitably 0.15 or less. In embodiments, the Ssk value is −0.8 or more atthis area scale, suitably −0.7 or more, suitably −0.6 or more, suitably−0.5 or more, suitably −0.4 or more, suitably −0.3 or more, suitably−0.2 or more, suitably −0.18 or more, suitably −0.15 or more.

In embodiments of any of the aspect or embodiment described herein, theimplant material at an area scale of 1 μm×1 μm comprises a mean Sskvalue of from −0.5 to 0.5, suitably −0.4 to 0.4, suitably −0.3 to 0.3,suitably −0.2 to 0.2, suitably −0.1 to 0.1, suitably −0.05 to 0.05, e.g.around zero. In embodiments, the Ssk value is 0.5 or less at this areascale, suitably 0.4 or less, suitably 0.3 or less, suitably 0.2 or less,suitably 0.1 or less, suitably 0.08 or less, suitably 0.06 or less. Inembodiments, the Ssk value is −0.5 or more at this area scale, suitably−0.4 or more, suitably −0.3 or more, suitably −0.2 or more, suitably−0.1 or more, suitably −0.08 or more, suitably −0.06 or more.

The invention thus provides an implant material according to any aspector embodiment herein wherein the surface at the respective area scaleshas a mean surface skewness Ssk value of:

-   -   a) from −1.0 to +1.0, suitably −0.2 to +0.2 at an area scale of        1 mm×1 mm; and/or    -   b) from −1.0 to +1.0, suitably −0.2 to +0.2 at an area scale of        90 μm×90 μm; and/or    -   c) from −0.7 to +0.7, suitably −0.2 to +0.2 at an area scale of        10 μm×10 μm; and/or    -   d) from −0.5 to +0.5, suitably −0.1 to 0.1 at an area scale of 1        μm×1 μm.

In preferred embodiments, the implant material has a mean surfaceskewness Ssk value of −0.2 to 0.2, suitably −0.15 to 1.5, suitably aboutzero at each respective area scale.

In embodiments, the implant surface Ssk value is substantially asdisclosed in any one or more of FIGS. 16A to 18D, and 26A to 41.

In embodiments of any of the aspects herein 40% to 60% of the surface atthe respective area scale consists of peaks, optionally wherein from 45%to 55% of the surface at the respective area scale consists of peaks,such as around 50% of the surface at the respective area scale consistsof peaks.

In embodiments of any of the aspects herein, from 40% to 60% of thesurface at the respective area scale consists of troughs, optionallywherein from 45% to 55% of the surface at the respective area scaleconsists of troughs, such as around 50%.

Embodiments have, in addition to the Ssk value(s) described above, oneor more of the Sa value(s) described herein and/or one or more of the Sqvalue(s) described herein and/or one or more of the Sz value(s)described herein and/or one or more of the kurtosis (Sku minus 3)value(s) described herein and/or one or more of the fractal dimension(FD) value(s) described herein and/or one or more of the surface linear(horizontal or vertical) auto correlation function (ACF) value(s)described herein.

Kurtosis and Mean Excess Kurtosis Value (Sku Minus 3)

The invention provides an implant material according to any aspect orembodiment disclosed herein wherein the surface is substantially free ofpeaks and troughs that deviate significantly from the mean surfaceheight roughness Sa value at the respective areas scale. In embodiments,the surface is substantially free of peaks or troughs which deviate fromthe mean surface height roughness Sa value by more than 200% of the meansurface height value at the respective area scales, suitably by morethan 150% of the mean surface height value at the respective areascales, such as by more than 100%. In this context, substantially freemeans that less than 2%, suitably less than 1%, preferably less than0.5%, of the implant material surface area consists of suchsignificantly deviating peaks.

In embodiments, the peak profile of the implant material surfaceaccording to any aspect herein is substantially rounded, squashed orflattened, e.g. compared to Mentor Siltex textured implant surfaces.

In embodiments of any of the aspect or embodiment described herein, theimplant material at an area scale of 1 mm×1 mm comprises a mean excesskurtosis value Sku minus 3 of from −1.5 to 1.5, suitably −1.0 to 1.0,suitably −0.7 to 0.7, suitably −0.5 to 0.5, suitably −0.3 to 0.3,suitably −0.2 to 0.2. In embodiments, the Sku minus 3 value is 1.5 orless at this area scale, suitably 1.0 or less, suitably 0.8 or less,suitably 0.7 or less, suitably 0.6 or less, suitably 0.5 or less,suitably 0.4 or less, suitably 0.3 or less, suitably 0.25 or less,suitably 0.2 or less, suitably 0.18 or less, suitably 0.15 or less. Inembodiments, the Skuu minus 3 value is −1.5 or more at this area scale,suitably −1.0 or more, suitably −0.8 or more, suitably −0.7 or more,suitably −0.6 or more, suitably −0.5 or more, suitably −0.4 or more,suitably −0.3 or more, suitably −0.2 or more, suitably −0.15 or more,suitably −0.1 or more, suitably −0.05 or more, suitably 0 or more.

In embodiments of any of the aspect or embodiment described herein, theimplant material at an area scale of 90 μm×90 μm comprises a mean excesskurtosis value Sku minus 3 of from −1.5 to 1.5, suitably −1.0 to 1.0,suitably −0.7 to 0.7, suitably −0.5 to 0.5, suitably −0.3 to 0.3,suitably −0.2 to 0.2. In embodiments, the Sku minus 3 value is 1.5 orless at this area scale, suitably 1.0 or less, suitably 0.8 or less,suitably 0.7 or less, suitably 0.6 or less, suitably 0.5 or less,suitably 0.4 or less, suitably 0.3 or less, suitably 0.25 or less,suitably 0.2 or less, suitably 0.18 or less, suitably 0.15 or less. Inembodiments, the Skuu minus 3 value is −1.5 or more at this area scale,suitably −1.0 or more, suitably −0.8 or more, suitably −0.7 or more,suitably −0.6 or more, suitably −0.5 or more, suitably −0.4 or more,suitably −0.3 or more, suitably −0.2 or more, suitably −0.15 or more,suitably −0.1 or more, suitably −0.05 or more, suitably 0 or more.

In embodiments of any of the aspect or embodiment described herein, theimplant material at an area scale of 10 μm×10 μm comprises a mean excesskurtosis value Sku minus 3 of from −1.5 to 1.5, suitably −1.0 to 1.0,suitably −0.7 to 0.7, suitably −0.5 to 0.5, suitably −0.3 to 0.3,suitably −0.2 to 0.2. In embodiments, the Sku minus 3 value is 1.5 orless at this area scale, suitably 1.0 or less, suitably 0.8 or less,suitably 0.7 or less, suitably 0.6 or less, suitably 0.5 or less,suitably 0.4 or less, suitably 0.3 or less, suitably 0.25 or less,suitably 0.2 or less, suitably 0.18 or less, suitably 0.15 or less. Inembodiments, the Skuu minus 3 value is −1.5 or more at this area scale,suitably −1.0 or more, suitably −0.8 or more, suitably −0.7 or more,suitably −0.6 or more, suitably −0.5 or more, suitably −0.4 or more,suitably −0.3 or more, suitably −0.2 or more, suitably −0.18 or more,suitably −0.16 or more, suitably −0.15 or more, suitably −0.1 or more,suitably −0.05 or more, suitably 0 or more.

In embodiments of any of the aspect or embodiment described herein, theimplant material at an area scale of 1 μm×1 μm comprises a mean excesskurtosis value Sku minus 3 of from −1.5 to 1.5, suitably −1.0 to 1.0,suitably −0.8 to 0.8, suitably −0.7 to 0.7, suitably −0.5 to 0.5,suitably −0.3 to 0.3, suitably −0.2 to 0.2. In embodiments, the Skuminus 3 value is 1.5 or less at this area scale, suitably 1.0 or less,suitably 0.8 or less, suitably 0.7 or less, suitably 0.6 or less,suitably 0.5 or less, suitably 0.4 or less, suitably 0.3 or less,suitably 0.25 or less, suitably 0.2 or less, suitably 0.18 or less,suitably 0.15 or less. In embodiments, the Skuu minus 3 value is −1.5 ormore at this area scale, suitably −1.0 or more, suitably −0.8 or more,suitably −0.7 or more, suitably −0.6 or more, suitably −0.5 or more,suitably −0.4 or more, suitably −0.3 or more, suitably −0.2 or more,suitably −0.18 or more, suitably −0.16 or more, suitably −0.15 or more,suitably −0.1 or more, suitably −0.05 or more, suitably 0 or more.

In embodiments of any of the aspects herein is provided an implantmaterial having a mean excess kurtosis value (Sku minus 3) of:

-   -   a) from −1.0 to +1.0, suitably −0.2 to +0.2 at an area scale of        1 mm×1 mm; and/or    -   b) from −1.0 to +1.0, suitably −0.2 to +0.2 at an area scale of        90 μm×90 μm; and/or    -   c) from −1.0 to +1.0, suitably −0.2 to +0.2 at an area scale of        10 μm×10 μm; and/or    -   d) from −1.5 to +1.5, suitably −0.1 to +0.1 at an area scale of        1 μm×1 μm.

In preferred embodiments, the implant material has a mean excesskurtosis value (Sku minus 3) of about zero at any one or more of therespective area scales.

In embodiments, the implant surface kurtosis value is substantially asdisclosed in any one or more of FIGS. 16A to 18D, and 26A to 41.

Embodiments have, in addition to the kurtosis (Sku minus 3) value(s)described above, one or more of the Sa value(s) described herein and/orone or more of the Sq value(s) described herein and/or one or more ofthe Sz value(s) described herein and/or one or more of the Ssk value(s)described herein and/or one or more of the fractal dimension (FD)value(s) described herein and/or one or more of the surface linear(horizontal or vertical) auto correlation function (ACF) value(s)described herein.

Fractal Dimension (FD)

In the aspects and embodiments herein, the implant material may have afractal dimension at the respective area scale of from 2.0 to 2.6.Suitably, the fractal dimension at the respective area scale is from 2.1to 2.5, suitably 2.2 to 2.4.

In embodiments of any of the aspect or embodiment described herein, theimplant material at an area scale of 1 mm×1 mm comprises a fractaldimension value FD of from 2.0 to 2.6, suitably 2.1 to 2.5, suitably 2.2to 2.4. In embodiments the FD value is 2.6 or less at this area scale,suitably 2.5 or less, suitably 2.45 or less, suitably 2.4 or less. Inembodiments the FD value is 2.0 or more at this area scale, suitably 2.1or more, suitably 2.2 or more, suitably 2.25 or more.

In embodiments of any of the aspect or embodiment described herein, theimplant material at an area scale of 90 μm×90 μm comprises a fractaldimension value FD of from 2.0 to 2.6, suitably 2.1 to 2.5, suitably 2.2to 2.4. In embodiments the FD value is 2.6 or less at this area scale,suitably 2.5 or less, suitably 2.45 or less, suitably 2.4 or less,suitably 2.35 or less. In embodiments the FD value is 2.0 or more atthis area scale, suitably 2.1 or more, suitably 2.2 or more, suitably2.25 or more.

In embodiments of any of the aspect or embodiment described herein, theimplant material at an area scale of 10 μm×10 μm comprises a fractaldimension value FD of from 2.0 to 2.6, suitably 2.1 to 2.5, suitably 2.2to 2.4. In embodiments the FD value is 2.6 or less at this area scale,suitably 2.5 or less, suitably 2.45 or less, suitably 2.4 or less,suitably 2.35 or less. In embodiments the FD value is 2.0 or more atthis area scale, suitably 2.1 or more, suitably 2.2 or more, suitably2.25 or more.

In embodiments of any of the aspect or embodiment described herein, theimplant material at an area scale of 1 μm×1 μm comprises a fractaldimension value FD of from 2.0 to 2.6, suitably 2.1 to 2.5, suitably 2.2to 2.4. In embodiments the FD value is 2.6 or less at this area scale,suitably 2.5 or less, suitably 2.45 or less, suitably 2.4 or less,suitably 2.35 or less. In embodiments the FD value is 2.0 or more atthis area scale, suitably 2.1 or more, suitably 2.2 or more, suitably2.25 or more.

In preferred embodiments, all of the respective area scales selectedfrom a) to d) have a fractal dimension as described above. Suchpreferred implant materials of the invention are therefore self-similarin the respective area dimensions (because the fractal dimension at eacharea scale is similar). This mimics the fractal arrangements of tissuesurfaces in the body, e.g. in the BM layer of the dermis of the skin andis proposed to provide a more natural biomimetic environment forcellular attachment and proliferation.

In preferred embodiments, the implant material according to any of theaspects herein has a fractal dimension substantially as defined in anyone or more of FIGS. 16A to 18D, and 24A to 28B.

Embodiments have, in addition to the Fractal Dimension described above,one or more of the Sa value(s) described herein and/or one or more ofthe Sq value(s) described herein and/or one or more of the Sz value(s)described herein and/or one or more of the Ssk value(s) described hereinand/or one or more of the kurtosis (Sku minus 3) value(s) describedherein and/or one or more of the surface linear (horizontal or vertical)auto correlation function (ACF) value(s) described herein.

Surface Linear (Horizontal or Vertical) Auto Correlation Function (ACF)

The correlation length is defined using a Gaussian fit in accordancewith Equation 1 to the first half of data points in the ACF. An exampleof such fit being illustrated in FIG. 42.

$\begin{matrix}\begin{matrix}{{{G\left( {\tau_{x},\tau_{y}} \right)} = {\int{\int_{- \infty}^{\infty}{z_{1}z_{2}{w\left( {z_{1},z_{2},\tau_{x},\tau_{y}} \right)}d\; z_{1}d\; z_{2}}}}}\ } \\{= {\lim\limits_{S\rightarrow\infty}{\frac{1}{S}{\int{\int_{S}{{\xi \left( {x_{1},y_{1}} \right)}{\xi \left( {{x_{1} + \tau_{x}},{y_{1} + \tau_{y}}} \right)}\ {dx}_{1}{dy}_{1}}}}}}}\end{matrix} & {{Equation}\mspace{14mu} 1}\end{matrix}$

where z₁ and z₂ are the values of heights at points (x₁, y₁), (x₂, y₂);furthermore, τ_(x)=x₁−x₂ and τ_(y)=y₁−y₂. The function w(z₁, z₂, τ_(x),τ_(y)) denotes the two-dimensional probability density of the randomfunction ξ(x, y) corresponding to points (x₁, y₁), (x₂, y₂), and thedistance between these points T. (Gwyddion user guide;http://gwyddion.net/documentation/user-guide-en/).

Reference herein to “Horizontal” and “Vertical” in the context of linearauto correlation function is a reference to the plan (planar) view, i.e.the X-axis (horizontal) and Y-axis (vertical).

In embodiments of any of the aspect or embodiment described herein, theimplant material at an area scale of 1 mm×1 mm has a linearautocorrelation length of from 20 μm to 200 μm, suitably 30 μm to 200μm, suitably 50 μm to 200 μm, suitably 60 μm to 190 μm. In embodimentsthe linear autocorrelation length is 200 μm or less at this area scale,suitably 190 μm or less, suitably 180 μm or less. In embodiments theautocorrelation length is 20 μm or more at this area scale, suitably 25μm or more, suitably 30 μm or more, suitably 35 μm or more.

In embodiments of any of the aspect or embodiment described herein, theimplant material at an area scale of 90 μm×90 μm has a linearautocorrelation length of from 3 μm to 20 μm, suitably 3 μm to 15 μm,suitably 3 μm to 13 μm, suitably 4 μm to 13 μm, suitably 5 μm to 11 μm,suitably 5 μm to 10 μm. In embodiments the linear autocorrelation lengthis 20 μm or less at this area scale, suitably 15 μm or less, suitably 13μm or less, suitably 12 μm or less, suitably 11 μm or less, suitably 10μm or less, suitably 9 μm or less. In embodiments the autocorrelationlength is 2 μm or more at this area scale, suitably 3 μm or more,suitably 4 μm or more, suitably 5 μm or more, suitably 5.5 μm or more,suitably 6 μm or more.

In embodiments of any of the aspect or embodiment described herein, theimplant material at an area scale of 10 μm×10 μm has a linearautocorrelation length of from 0.5 μm to 2.5 μm, suitably 0.5 μm to 2μm, suitably 0.6 μm to 2 μm, suitably 0.6 μm to 1.8 μm, suitably 0.7 μmto 1.8 μm. In embodiments the linear autocorrelation length is 2.5 μm orless at this area scale, suitably 2 μm or less, suitably 1.9 μm or less,suitably 1.8 μm or less, suitably 1.75 μm or less, suitably 1.7 μm orless, suitably 1.65 μm or less, suitably 1.6 or less, suitably 1.6 orless, suitably 1.55 μm or less, suitably 1.5 μm or less. In embodimentsthe autocorrelation length is 0.5 μm or more at this area scale,suitably 0.55 μm or more, suitably 0.6 μm or more, suitably 0.65 μm ormore, suitably 0.7 μm or more, suitably 0.75 μm or more, suitably 0.8 μmor more, suitably 0.85 μm or more.

In embodiments of any of the aspect or embodiment described herein, theimplant material at an area scale of 1 μm×1 μm has a linearautocorrelation length of from 40 nm to 200 nm, suitably 20 nm to 180nm, suitably 40 nm to 180 nm, suitably 60 nm to 180 nm, suitably 50 nmto 150 nm, suitably 60 nm to 130 nm. In embodiments the linearautocorrelation length is 200 nm or less at this area scale, suitably190 nm or less, suitably 180 nm or less, suitably 175 nm or less,suitably 170 nm or less, suitably 165 nm or less, suitably 160 nm orless, suitably 155 nm or less, suitably 155 nm or less, suitably 150 nmor less, suitably 145 nm or less, suitably 140 nm or less, suitably 135nm or less, suitably 130 nm or less. In embodiments the autocorrelationlength is 40 nm or more at this area scale, suitably 45 nm or more,suitably 50 nm or more, suitably 55 nm or more, suitably 60 nm or more,suitably 65 nm or more, suitably 70 nm or more, suitably 75 nm or more,suitably 80 nm or more, suitably 85 nm or more.

In embodiments, the implant material has a surface having a linearcorrelation length of:

-   -   a) from 20 μm to 200 μm, suitably 60 μm to 190 μm, at an area        scale of 1 mm×1 mm; and/or    -   b) from 3 μm to 15 μm, suitably 5 μm to 13 μm at an area scale        of 90 μm×90 μm;

and/or

-   -   c) from 0.5 μm to 2.5 μm, suitably 0.7 μm to 2 μm at an area        scale of 10 μm×10 μm and/or    -   d) from 40 nm to 150 nm, suitably 60 nm to 130 nm at an area        scale of 1×1 μm.

In embodiments, the ranges and values given above are the horizontalauto correlation length. Thus, embodiments have a horizontal autocorrelation length satisfying the ranges given above.

In embodiments, the values given above are the vertical auto correlationlength. Thus, embodiments have a vertical auto correlation lengthsatisfying the ranges given above.

In embodiments, the values given above are for both the horizontal autocorrelation length and the vertical auto correlation length. Thus,embodiments have a horizontal auto correlation length satisfying theranges given above and a vertical auto correlation length satisfying theranges given above.

In embodiments, the horizontal auto correlation length is:

-   -   a) from 20 μm to 200 μm, suitably 60 μm to 190 μm, at an area        scale of 1 mm×1 mm; and/or    -   b) from 3 μm to 15 μm, suitably 5 μm to 13 μm at an area scale        of 90 μm×90 μm; and/or    -   c) from 0.5 μm to 2.5 μm, suitably 0.7 μm to 2.0 μm at an area        scale of 10 μm×10 μm and/or    -   d) from 40 nm to 150 nm, suitably 60 nm to 130 nm at an area        scale of 1×1 μm.

In embodiments, the vertical autocorrelation length is:

-   -   a) from 20 μm to 200 μm, suitably 60 μm to 190 μm, at an area        scale of 1 mm×1 mm; and/or    -   b) from 3 μm to 15 μm, suitably 5 μm to 13 μm at an area scale        of 90 μm×90 μm; and/or    -   c) from 0.5 μm to 2.5 μm, suitably 0.7 μm to 2.0 μm at an area        scale of 10 μm×10 μm and/or    -   d) from 40 nm to 150 nm, suitably 60 nm to 130 nm at an area        scale of 1×1 μm.

In embodiments of any of the aspects herein, the implant material at anarea scale of 1 mm×1 mm has a minimum in the ACF from 200 to 600 μm andoptionally a second maximum from 500 to 850 μm.

In embodiments of any of the aspects herein, the implant material at anarea scale of 90 μm×90 μm has a minimum in the ACF from 15 to 50 μm andoptionally a second maximum from 50 to 85 μm.

In embodiments of any of the aspects herein, the implant material at anarea scale of 10 μm×10 μm has a minimum in the ACF from 1 to 6 μm andoptionally a second maximum from 6 to 9 μm.

In embodiments of any of the aspects herein, the implant material at anarea scale of 1×1 μm may have a minimum in the ACF from 0.1 to 0.6 μmand optionally a second maximum from 0.6 to 0.9 μm.

In embodiments, the implant material according to any of the aspectsherein has a first minimum and optionally a second maximum in the ACFsubstantially as described herein in FIG. 42.

In embodiments at least half, preferably more than half, more preferablysubstantially all, most preferably all of the peaks and troughs of thesurfaces of the invention have a gradually sloped gradient. In preferredembodiments, the textured surfaces of the invention are substantiallyfree from steep slopes or side-walls (such as slopes having an inclineof around 60-90%). This feature has the advantage of improving cellularmobility and survival by preventing cells becoming trapped withintroughs in the surface on implantation.

Preferably the surfaces of the invention are substantially free,suitably free from open cell textures.

Implant Material

In embodiments of any of the aspects herein the implant materialcomprises, suitably comprises as a major component (e.g. at least 50 wt% of the total weight of the implant material, preferably at least 60 wt%, more preferably at least 70 wt %, more preferably at least 80 wt %,more preferably at least 90 wt %) in embodiments consist substantiallyof, in typical embodiments consist of, a suitable biocompatiblematerial.

Suitably the material is capable of being shaped, e.g. by castingetching and/or moulding into a textured surface. Suitably, the materialmay comprise suitably comprises as a major component (e.g. at least 50wt % of the total weight of the implant material, preferably at least 60wt %, more preferably at least 70 wt %, more preferably at least 80 wt%, more preferably at least 90 wt %, more preferably at least 95 wt %,more preferably at least 99 wt %) in embodiments consist substantiallyof, typically consist of, a biocompatible synthetic polymer, suitably anorgano-silicon polymer, preferably a silicone, and more preferablypolydimethylsiloxane (PDMS).

It is particularly preferred that the surface of the implant materialfor which surface roughness parameters are specified herein (i.e. thesurface intended to contact the patient's tissue, i.e. thetissue-engaging surface) comprises the above-mentioned biocompatiblesynthetic polymer. Indeed, as noted above, suitably the surface consistssubstantially of an organo-silicon polymer, preferably PDMA. Thus, inembodiments, the surface (tissue-engaging surface) is a texturedorgano-silicon surface, the texture being as described herein.

Suitably the composition of the implant material is substantiallyhomogeneous, especially in a depth direction from the surface(tissue-engaging surface) into the bulk material.

The implant material suitably forms at least part of the surface layerof the relevant implant. Thus, surfaces of implants of the invention maypartly comprise conventional implant surfaces as well as the novel andadvantageous surfaces described herein. In embodiments, the implantmaterial surfaces of the invention described herein forms at least half,in suitable embodiments more than half, preferably substantially all(e.g. at least 90%, 95%, 98% or 99% by area of the implant surface) ofthe tissue contacting surface of the implant, such as wherein the tissuecontacting surface of the implant consists of said implant material. Thematerial comprising the surfaces of the invention may be different toother materials in the implant or may be the same. Thus the implant maycomprise an underlayer layer of the same of different material to theimplant surface layer of the invention.

The implant may be any suitable implant capable of insertion into apatient, preferably a prosthetic implant, optionally an implant forinternal insertion beneath the skin surface of a patient, morepreferably a breast implant.

The implant materials of the present invention are preferably configuredso as to be inserted subcutaneously within a patient or may beadministered externally. Preferably the implant is administered (isintended to be located) internally, e.g. subcutaneously.

Templates

In a further aspect of the invention is provided a template for use inpreparing an implant material according to any aspect or embodimentherein. Suitably, said template comprises a textured surface asdescribed according to any aspect or embodiment herein, or a negative(e.g. an inverse cast) of a textured surface as described herein. Thetemplate may typically comprise the 3-dimensional information, i.e.X,Y,Z information, corresponding to the implant material surface of theinvention as defined according to any aspect and embodiment herein. Inembodiments, the template is a stamp or mould, e.g. a stamp forimprinting a surface texture of the invention onto an implant surface ormoulding the implant surface, optionally wherein the stamp or mould is asilicone stamp or mould. Thus, a surface may be stamped or moulded anumber of times to provide an implant material having a surface asdefined above. In embodiments, the template itself is a mould. The useof moulds is beneficial as they can be used to manufacture a largenumber of implants quickly.

Methods

In an aspect of the invention is provided a method of preparing animplant material having a textured surface comprising the steps ofacquiring spatial data in the X, Y and Z dimensions (i.e.three-dimensional spatial data) from a tissue surface and using saidspatial data to create the textured surface of the implant.

Suitably, the use of the spatial data further comprises the step ofprocessing the spatial data and using the processed data to create thetextured surface of the implant. Preferably the textured surface is anirregular textured surface.

The inventors thus propose the acquisition of 3D image/topography datacorresponding to a tissue surface for reproduction on (formation of) animplant surface. This approach represents a considerable departure fromconventional approaches to texturising implant surfaces, which arelargely based on trial and error application of crude and oftenirreproducible methods which do not provide suitable control of theimplant surfaces produced (e.g. by making open cell foam or bytexturising using salt methods).

In embodiments, the step of acquiring the spatial X,Y,Z data isperformed by any suitable contact or non-contact profilometer, suitablyby atomic force microscopy, 3D laser scanner or optical profiler.Preferably, atomic force microscopy is used.

In embodiments, the step of creating the textured surface using thespatial X,Y,Z data includes three dimensional printing orphotolithography or E-beam lithography, particularly opticalphotolithography, e.g. UV lithography. The level of resolution which canbe achieved in the implant surfaces using a lithography method of theinvention (see, e.g. Example 1) may be adjusted by a number of steps inthe manufacture method. For instance, AFM scanners typically do notallow measurement of multiple 10's of microns in vertical scale. Thegrayscale lithography method as disclosed herein (see Example 1) iscapable of reproducing lateral resolution of ˜500 nm and thus isexcellent as a method of reproducing micro scale and larger nano-scalefeatures in surfaces of the present invention but excluding nano-scalefeatures and long distance waviness features, etc. Thus, in anembodiment, the method includes the step of processing the 3D data(spatial X,Y,Z) by converting, suitably digitally converting therespective data to a form of data that can be read by a masklesslithography system. In an embodiment, the processing step includesformation of a two or more 8 bit (or optionally 16 bit) grayscale imagewherein the 256 (e.g. or optionally 65536) different grayscaleintensities corresponds to changes in vertical height of a measuredsurface. Alternatively or additionally, the processing step includesjoining two or more grayscale images (maps) to form a mosaic montage ofsurface images prior to applying the image to a surface, for exampleprior to assigning a number of radiation doses on every pixel and thuscontrolling the exposure of the photoresist.

Use of such methods thus allows the production of controlled irregularsurface features in an implant surface, which are, based on thereproduction of surface roughness features taken from a natural cellularenvironment and not from surfaces manufactured by the crude anduncontrolled ways reported in prior art. The method is more versatilethan prior art methods and adaptation of the digital X,Y,Z informationcan provide not only the cell topography itself, but a variety ofsurface topographies using the tissue surface features as the originalinspiration. Processing and manipulation of the surface data during thelithography or printing allows for reproduction of an endless range ofsurface designs. FIG. 25 describes a surface produced according to thegrayscale method compared with natural material to show thesimilarities.

Use of Electron Beam (E-beam) Lithography may allow the reproduction offeatures that are <50 nm in lateral resolution. Thus, in an embodiment,the process of forming the surface of the invention from using thespatial X,Y,Z data includes using Electron Beam (E-beam) Lithography.

In embodiments, the method further comprises using the spatial X,Y,Zdata to expose a photoresist (for example an E-beam photoresist)comprising the respective X,Y,Z information.

The method suitably includes use of the exposed and developedphotoresist (for example an E-beam resist) to form the textured surface.The step of using the exposed and developed photoresist to transfer thetextured surface onto a template may optionally comprise using anetching method, optionally deep reactive ion etching.

Embodiments of the method include use of the spatial X,Y,Z data toexpose the photoresist and/or an e-beam resist comprising using thespatial X,Y,Z data to instruct the delivery of varying doses ofradiation to a photoresist and/or E-beam resist surface so as to exposea photoresist and/or E-beam resist comprising the respective X,Y,Zinformation. Usually photolithography methods for preparing 3D featuresin objects (such as commonly used in the semiconductor industry) use agraded photomask to control the relative intensity of radiation receivedby various parts of the photoresist during the photolithography step.However, it is expensive and time-consuming to prepare such photomasksand once made, they cannot be varied and must be used to make a range ofidentical patterns. To the contrary, the use of the X,Y,Z data (e.g. thecolour or grayscale depiction of peak-trough height) to control therelative intensity of radiation received at a given point of thephotoresist (such as by using laserwriter configured to read suchgrayscale data) can advantageously allow for the exposure of aphotoresist having, after development, the surface features directlyrather than using a photomask. In other words, in embodiments, thelithography method is a maskless lithography method.

In embodiments, the step of preparing the photoresist includesincreasing or decreasing the scale of the original X, Y and/or Zparameters for reproduction in the photoresist. This may be usedadvantageously if the photoresist needs to be thinner in the verticaldirection that the vertical features of the surface being reproduced.The features may this be scaled up again during another step, such asusing etching, e.g. deep reactive ion etching.

In another aspect is a method of preparing an implant material having atextured surface comprising the step of making a cast of a texturedtissue surface, the cast containing spatial data representing the X,Yand Z dimensions and using said cast to make the textured implantmaterial.

Casting of the tissue surface (see Example 2 and FIGS. 27A-27C) has theadvantage that it allows for precise replication of the topographicalfeatures of tissue samples, such as human-derived decellularised dermis,e.g. acellular dermis (ADM). Moreover, casting method allows replicationof the full range of features of the tissue surface topography and thusis not limited by the inherent resolution of imaging and/or lithographymethods (see FIGS. 27A-27C for a comparison of natural ADM BM tissuesurface and ADM BM C as cast according to the method above showing aclose reproduction of the natural features in the cast implant surface).

Method of Applying Texture to the Surfaces of the Invention

In embodiments, the method comprises the step of preparing said texturedimplant material surface by etching, stamping or moulding. Inembodiments, the method comprises the step of preparing said texturedimplant material surface by etching. In embodiments the method comprisesthe step of preparing said textured implant material surface bystamping, optionally multiple stamping of a single surface to produce atextured surface having a number of stamped irregular textured regions,e.g. wherein the stamped images cover at least half, suitably more thanhalf, and in embodiments substantially all of the implant surfaceconfigured to contact a patient's tissue when inserted. In embodimentsthe method comprises the step of preparing said textured implantmaterial surface by moulding.

In embodiments the implant material prepared by said method is animplant material as described in any one of the aspects and embodimentsof the invention described herein.

Data Set

In an aspect of the invention is the use of spatial data representingthe X, Y and Z dimensions acquired from a tissue surface in a method ofpreparing a textured material or a photoresist for use in preparing atextured material. In embodiments, the textured material is a texturedimplant material as described herein or a template as described herein.

In an aspect of the invention is a method of processing and/or modifyingspatial data in the X, Y and Z dimensions, suitably so as to provide adata set capable of being used by a printer, for example a laser writeror 3D printer.

In embodiments, the use includes wherein the spatial data acquired fromthe tissue surface is processed before use in said method ofpreparation.

In an aspect of the invention is spatial data in the X, Y and Zdimensions acquired from a tissue surface.

In an aspect of the invention is a data carrier, suitably a computerreadable data carrier, comprising spatial data as defined herein.

Tissue

In the above methods of uses, the tissue (i.e. the tissue from whichspatial data is the X, Y and Z dimensions has been obtained or isrepresentative of) may be any suitable body tissue, preferably internalbody tissues. In embodiments, the tissue is dermal tissue, optionallywherein said dermal tissue is selected from the basement membrane of theskin, the papillary dermis layer of the skin; or the basement membranesuperimposed on the papillary dermis of the skin, preferably wherein therespective dermal tissue is the BM surface layer of acellular dermalmatrix.

In embodiments the tissue is acellular dermal matrix, optionally whereinthe acellular dermal matrix is selected from the basement membrane ofacellular dermis, the papillary dermis layer of acellular dermis or thebasement membrane overlying papillary dermis layer in the acellulardermis.

The basement membrane (BM) is a thin layer (20-100 nm thick) ofcollagens (IV and VII), glycoproteins (entactin and perlecan) andproteoglycans (heparan sulfate and glycosaminoglycans) that separateepidermis and dermis within skin and epithelial and underlying layerswithin other tissues/organs throughout the body. Its function is organspecific and therefore its composition and morphology throughout thebody reflects this. The BM in skin is a fusion of the basal lamina (BL)and lamina reticularis (LR) through anchoring fibrils such as collagenVII. The basal lamina can be separated further into 2 distinct layers:lamina lucida (LL) and lamina densa (LD). Within skin, the LD and LR(deepest layers of BM) provide spatial and mechanical cues forfibroblasts to attach to and perform their individual functions. Itdirects cell differentiation and cell process through a synergisingeffect of attached growth factors, topographical and mechanical(stiffness) cues.

The BM overlying PD within skin contains an abundance of specificallyperiodic and specially orientated motifs for cell attachment, and/orproliferation/differentiation. The topographical features of the BM arenanometre size whilst the underlying PD is on a micron scale; workingsynergistically. These topographical cues are specific to the cellsinteracting with them and appear to encourage normal cellular processes.Therefore, a fibroblast in vivo attached to the BM will not be in anunfamiliar or stressed state. Accordingly, in normal circumstances inthe body, cells are not inflammatory or hyperproliferative and a foreignbody reaction does not ensue. Further, collagen type IV is necessary fornormal mammary epithelial cell attachment in vivo and while notessential for normal mammary fibroblast attachment it may still promoteeffective cell attachment. As mentioned previously, collagen type IV isa component of the basement membrane and therefore replication of BMtopography into silicone may promote mammary epithelial and fibroblastcell attachment and proliferation in vivo.

Through mimicking the topographical cues of BM/PD onto the surface of asilicone implant, cells that encounter it attach and stabilize withoutbecoming stressed and transforming into a pro-inflammatory/fibroticphenotype resulting in the initiation of chronic inflammation andfibrosis around the implant through attraction and activation ofneutrophils and macrophages (see cellular response data and discussion).Consequently, it is thought that the extent of the foreign body reactionand subsequent capsular contracture formation would be potentiallyaverted.

Whilst it is understood that natural BM/PD may be able to effect suchfunctions in the body, it is entirely surprising that the excellentresults achieved using the fabricated and cast materials prepared wouldshow the excellent results observed when the 3D topographic featureswere reproduced in silicone implant surfaces as discussed in theexamples section. The investigation by the present inventors hastherefore led to a ground-breaking innovation that has the potential tohave significant impact on 3D surface design for implants, particularlyin the breast implant field with the effect of reducing problemsassociated with this growing demand for this surgical procedure onmedical and/or cosmetic grounds.

As discussed, present methods for forming irregular implant surfacestypically rely on crude and inherently unpredictable processes. Suchmethods thus provide non-reproducible surfaces that may differsignificantly from batch to batch, leading to potentially unreliableresults. It is however desirable to be able to control the surfacefeatures with a high degree of accuracy, particularly in the case ofprosthetic implants such as breast implants, where differences in microand nano features have been proven to play an important role in cellularinteraction, biocompatibility and capsular contraction. The presentinvention thus addresses the need for methods which provide control ofirregular surface features with a higher degree of accuracy andreproducibility and/or which provide a higher degree of flexibility forproducing different designs.

Further Aspects

In a further aspect is provided an implant material comprising atextured surface as prepared by a method as defined according to anyaspect or embodiment herein.

Also provided is a template for use in preparing an implant material ofthe invention as described herein, said template having textured surfaceparameters as defined according to any previous claim, or a negative ofsaid textured surface parameters, optionally wherein the template is amould or stamp, such as defined above.

The invention also provides the use of a template as described accordingto any embodiment above in a method of making a textured implantmaterial. Typically the template is a silicone template, most preferablyPDMS.

A cosmetic method comprising the step of inserting an implant materialas described in any of the aspects and embodiments of the inventiondisclosed herein subcutaneously in a patient. Suitably said method is soas to provide minimal or no capsular contraction and/or cellularimmunogenic response. Furthermore, in embodiments the method is forreconstructions of the breast.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. an implant “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

The use of the term “irregular” in the context of the surfaces of thepresent invention will be well understood by the skilled person.Suitably, the term “irregular” in the context of the surfaces of thepresent invention refers to surface areas which are devoid of regulargeometric patterns (such as repeating patterns), such as at the relevantmacro, micro and/or nano scales (such as at the 1 cm×1 cm, 1 mm×1 mm,100 micron×100 micron and/or at sub-micron level). The term “irregular”in the context of the surfaces of the present invention thus includessurfaces which appear to be disordered.

It will be appreciated on reading the present application that thesurface of implants prepared according to the present invention may beformed by use of a stamp having an irregular textured surface whichimparts its irregular surface topography to the implant on stamping. Thestamp may thus be used repeatedly over the surface of the implant toultimately provide up to complete surface coverage consisting of thesubstantially repeated irregular surface imprinted by the stamp. It isthus intended that the term “irregular” within the meaning of thepresent invention includes surfaces which have more than one, such as aplurality of repeating areas of such irregular surface topography.

The terms “ACD” and “ADM” are used interchangeably and refer toacellular (decellularised) dermal matrix.

DESCRIPTION OF FIGURES

FIG. 1 depicts a grayscale montage of AFM images of ADM BM as producedby the fabrication method of the invention described in example 1.

FIG. 2 depicts breast derived fibroblast (BDF) cellular attachment dataover 1 to 6 h on ADM BM C and ADM BM F surfaces according to theinvention (as prepared according to the casting and fabrication methodsof the invention respectively—see examples 1 and 2) as compared togrowth on comparative smooth and textured implants, TCP and collagen.

FIG. 3 depicts breast derived fibroblast (BDF) cellular proliferationdata for up to 1 week on ADM BM C and ADM BM F surfaces according to theinvention (as prepared according to the casting and fabrication methodsof the invention respectively—see examples 1 and 2) as compared togrowth on comparative smooth and textured implants, TCP and collagen.

FIG. 4 depicts breast derived fibroblast (BDF) cell survival data for upto 1 week on ADM BM C and ADM BM F surfaces according to the invention(as prepared according to the casting and fabrication methods of theinvention respectively—see examples 1 and 2) as compared to growth oncomparative smooth and textured implants, TCP and collagen.

FIG. 5 depicts QRT-PCR data showing changes in PCNA (proliferating cellnuclear antigen) gene expression of breast derived fibroblast (BDF's)grown on ADM BM C and ADM BM F surfaces according to the invention (asprepared according to the casting and fabrication methods of theinvention respectively—see examples 1 and 2) as compared to growth oncomparative smooth and textured implants, TCP and collagen for 96 h.

FIG. 6 depicts QRT-PCR data showing changes in vinculin gene expressionof breast derived fibroblast (BDF's) grown on ADM BM C and ADM BM Fsurfaces according to the invention (as prepared according to thecasting and fabrication methods of the invention respectively) ascompared to growth on comparative smooth and textured implants, TCP andcollagen for 96 h.

FIG. 7 depicts QRT-PCR data showing changes in IL8 (Interleukin 8) geneexpression of breast-derived fibroblast (BDF's) grown on ADM BM C andADM BM F surfaces according to the invention (as prepared according tothe casting and fabrication methods of the invention respectively—seeexamples 1 and 2) as compared to growth on comparative smooth andtextured implants, TCP and collagen for 96 h.

FIG. 8 depicts Quantitative Real Time Polymerase Chain Reaction(QRT-PCR) data showing changes in TNF-alpha (Tumour necrosis factoralpha) gene expression of breast derived fibroblast (BDF's) grown on ADMBM C and ADM BM F surfaces according to the invention (as preparedaccording to the casting and fabrication methods of the inventionrespectively—see examples 1 and 2) as compared to growth on comparativesmooth and textured implants, TCP and collagen for 96 h.

FIGS. 9A-9B depict immunofluorescence cell morphology and phenotype datafor BDF's grown on ADM BM C (90×90 μm) according to the invention (asprepared according to the casting method of the invention). The imagesshow staining of F-Actin (red in original colour images), vinculin(green in original colour images) and Dapi (blue in original colourimages). White circles highlight vinculin staining and focal adhesionformation.

FIGS. 10A-10B depict immunofluorescence cell morphology and phenotypedata for BDF's grown on comparative smooth implants (Mentor smooth). Theimages show staining of F-Actin (red in original colour images),vinculin (green in original colour images) and Dapi (blue in originalcolour images).

FIG. 11 depicts immunofluorescence cell morphology and phenotype datafor BDF's grown on comparative textured implants (Mentor Siltex). Theimages show staining of F-Actin (red in original colour images),vinculin (green in original colour images) and Dapi (blue in originalcolour images). White boxes highlight vinculin staining and focaladhesion formation.

FIG. 12 depicts immunofluorescence cell morphology and phenotype datafor BDF's grown on tissue culture plastic (TCP). The images showstaining of F-Actin (red in original colour images), vinculin (green inoriginal colour images) and Dapi (blue in original colour images). Whitecircles highlight vinculin staining and focal adhesion formation.

FIGS. 13A-13B depict immunofluorescence cell morphology and phenotypedata for BDF's grown on collagen. The images show staining of F-Actin(red in original colour images), vinculin (green in original colourimages) Dapi (blue in original colour images). White circles highlightvinculin staining and focal adhesion formation.

FIG. 14 depicts SEM image data showing BDF growth on ADM BM C implantsurface according to the invention after 6 hours. BDF's have clearlyattached and are beginning to spread on the surface (white circleindicates a BDF which is displaying typical fibroblast spreadmorphology).

FIG. 15 depicts SEM images showing BDF growth on ADM BM C according tothe invention after 24 hours (A) and 48 hours (B-D). It is clear to seethe spread morphology of the cells on the ADM surface (white circleindicates a BDF which is displaying typical fibroblast spreadmorphology).

FIGS. 16A-16D depict Atomic Force Microscope (AFM) data for an imagescan size of 90×90 um for ADM BM surface topography according to theinvention. FIG. 16A shows the 2D planar surface depiction image of theAFM data, FIG. 16B shows the corresponding 3D image, FIG. 16C shows theAFM line profile data (in the vertical and horizontal direction)corresponding to the diagonal line shown in FIG. 16D (FIG. 16D alsoshowing the respective ISO 25178 areal data for the surface of FIG.16A).

FIGS. 17A-170 depict Atomic Force Microscope (AFM) data for an imagescan size of 10×10 um for ADM BM surface topography according to theinvention. FIG. 17A shows the 2D planar surface depiction image of theAFM data, FIG. 17B shows the corresponding 3D image, FIG. 17C shows theAFM line profile data (in the vertical and horizontal direction)corresponding to the diagonal line shown in FIG. 17D (FIG. 17D alsoshowing the respective ISO 25178 areal data for the surface of FIG.17A).

FIGS. 18A-18D depict Atomic Force Microscope (AFM) data for an imagescan size of 1×1 um for an ADM BM surface topography according to theinvention. FIG. 18A shows the 2D planar surface depiction image of theAFM data, FIG. 18B shows the corresponding 3D image, FIG. 18C shows theAFM line profile data (in the vertical and horizontal direction)corresponding to the diagonal line shown in FIG. 18D (FIG. 18D alsoshowing the respective ISO 25178 areal data for the surface of FIG.18A).

FIGS. 19A-19D depict Atomic Force Microscope (AFM) data for an imagescan size of 90×90 um for comparative smooth implant surface topography(Mentor Smooth). FIG. 19A shows the 2D planar surface depiction image ofthe AFM data, FIG. 19B shows the corresponding 3D image, FIG. 19C showsthe AFM line profile data (in the vertical and horizontal direction)corresponding to the diagonal line shown in FIG. 19D (FIG. 19D alsoshowing the respective ISO 25178 areal data for the surface of FIG.19A).

FIGS. 20A-20D depict 3D laser scanner data for an image scan size of 1mm×1 mm for comparative textured implant surface topography (MentorSiltex). FIG. 20A shows the 2D planar surface depiction image of the AFMdata, FIG. 20B shows the corresponding 3D image, FIG. 20C shows the AFMline profile data (in the vertical and horizontal direction)corresponding to the diagonal line shown in FIG. 20D (FIG. 20D alsoshowing the respective ISO 25178 areal data for the surface of FIG.20A).

FIGS. 21A-21D depict 3D laser scanner data for an image scan size of 100micron×100 micron for a comparative textured implant surface topography(Mentor Siltex). FIG. 21A shows the 2D planar surface depiction image ofthe AFM data, FIG. 21B shows the corresponding 3D image, FIG. 21C showsthe AFM line profile data (in the vertical and horizontal direction)corresponding to the diagonal line shown in FIG. 21D (FIG. 21D alsoshowing the respective ISO 25178 areal data for the surface of FIG.21A).

FIGS. 22A-22C depict Optical Microscopy images along with thecorresponding 2D (vertical vs lateral) profile measured on aprofilometer for ADM BM F surface topography fabricated according to theinvention (FIG. 22A), comparative smooth implants (Mentor Smooth, FIG.22B) and comparative textured (Mentor Siltex, FIG. 22C).

FIGS. 23A-22L depict SEM image data surface topographies at a variety ofarea scales for ADM BM according to the invention (FIG. 23A: 1 mm scaleleft, 500 μm scale right; FIG. 23B: 100 μm scale left, 50 μm scaleright; and FIG. 23C: 20 μm scale left and 10 um scale right); naturalADM BM surface topography (FIG. 23D: 1 mm scale left, 500 μm scaleright; FIG. 23E: 100 μm scale left, 50 um scale right; and FIG. 23F: 20um scale left and 10 μm scale right); comparative smooth implant (MentorSmooth) surface topography (FIG. 23G: 100 μm scale; FIG. 23H: 50 μmscale; and FIG. 23I: 1 μm scale); and comparative textured implant(Mentor Siltex) surface topography (FIG. 23J: 1 mm scale; FIG. 23K: 500μm scale; and FIG. 23L: 1 μm scale bottom).

FIGS. 24A-24B depict ISO 25178 areal surface roughness measurementscalculated as described herein describing (FIG. 24A) the 3D surfacetexture of ADM BM surface according to the invention compared tocommercially available smooth (Mentor smooth) and (FIG. 24B) textured(Mentor Siltex) implants.

FIG. 25 Comparison of roughness values of Native ADM BM in comparison toADM BM PDMS F and ADM BM PDMS C at different length scales.

FIGS. 26A-26C depict Atomic Force Microsope (AFM) data for an image scansize of 90×90 μm for natural ADM BM surface topography and ADM BM Fsurface topography manufactured according to the maskless grayscalelithography method of the invention as described herein.

FIG. 26A shows the 2D image of each surface (natural ADM BM at left, andADM BM PDMS F at right), FIG. 26B shows the 3D image, and FIG. 26C showsthe 2D profile data (vertical vs lateral dimension).

FIGS. 27A-27C depict Atomic Force Microsope (AFM) data for an image scansize of 90×90 μm for natural ADM BM surface topography and ADM BM Csurface topography manufactured according to the casting method of theinvention as described herein. FIG. 27A shows the 2D image of eachsurface (natural ADM BM at left, and ADM BM PDMS C at right), FIG. 27Bshows the 3D image, and FIG. 27C shows the 2D profile data (vertical vslateral dimension).

FIGS. 28A-28B show a comparison of native ADM BM (FIG. 28A) to ADM BMPDMS F (FIG. 28B) at 90×90 μm.

FIGS. 29A-29B show a comparison of native ADM BM (FIG. 29A) to ADM BMPDMS F (FIG. 29B) at 90×90 μm—Autocorrelation lengths.

FIGS. 30A-30B show a comparison of native ADM BM (FIG. 30A) to ADM BMPDMS F (FIG. 30B) at 10×10 μm.

FIG. 31 shows ADM BM PDMS F at 10×10 μm—Autocorrelation lengths.

FIGS. 32A-32B show a comparison of native ADM BM (FIG. 32A) to ADM BMPDMS F (FIG. 32B) at 1×1 μm.

FIGS. 33A-33B show a comparison of native ADM BM (FIG. 33A) to ADM BMPDMS F (FIG. 33B) at 1×1 μm—Autocorrelation lengths.

FIGS. 34A-34B show a comparison of native ADM BM (FIG. 34A) to ADM BMPDMS C (FIG. 34B) at 90×90 μm.

FIGS. 35A-35B show a comparison of native ADM BM (FIG. 35A) to ADM BMPDMS C (FIG. 35B) at 90×90 μm—Autocorrelation lengths.

FIGS. 36A-356 show a comparison of native ADM BM (FIG. 36A) to ADM BMPDMS C (FIG. 36B) at 10×10 μm.

FIGS. 37A-37B show a comparison of native ADM BM (FIG. 37A) to ADM BMPDMS C (FIG. 37B) at 10)(10 μm—Autocorrelation lengths.

FIGS. 38A-38B show a comparison of native ADM BM (FIG. 38A) to ADM BMPDMS C (FIG. 38B) at 1×1 μm.

FIGS. 39A-39B show a comparison of native ADM BM (FIG. 39A) to ADM BMPDMS C (FIG. 39B) at 1×1 μm—Autocorrelation lengths.

FIG. 40 shows a comparison of native ADM BM to ADM BM PDMS F AFM and SEMimages at 90×90 um.

FIG. 41 shows SEM images of native ADM BM, ADM BM PDMS C and ADM BM PDMSF at different magnifications.

FIG. 42 depicts a horizontal ACF function with Gaussian fit. The firstminimum and second minimum are indicated on the ACF data line.

FIG. 43 shows autocorrelation length data for native ADM BM, ADM BM PDMSF and ADM BM PDMS C at different length scales.

The invention is described in more detail by way of example only withreference to the following Examples and experimental procedures.

General Methods

Isolation of Acellular Dermis (Acd) from Cadaver Skin

ADM is soft and buff white in colour. Donors have been screened forpotential infective organisms and tissue has been deemed to be suitablefor transplantation based on the results of stringent donor testing. TheADM sample was completely aseptically decellularised and freeze driedwhile preserving biological components and morphology of dermal matrix.

Storage and Preparation of Adm for Imaging

Method A: Samples were stored at −20° C. and thawed on ice whenrequired. A 5 cm×5 cm of human acellular dermal matrix was removed fromthe packaging and placed in a petri dish. A section of ADM,approximately 2×2 cm, was excised from the sample and spread out inanother petri dish. This sample was fixed in 2% glutaraldehyde, in 0.1 MpH 7.4 phosphate buffer solution at 8° C. for 12 hours. The tissue wasthen washed with phosphate buffer solution (PBS)×3 to remove anyresidual glutaraldehyde. The tissue was then dehydrated using increasingconcentrations of intermediary fluid (ethanol) and was kept in 100%ethanol until it was ready for critical point drying (CPD). After CPDthe sample was ready for imaging.

Method B: The ADM was removed from storage in −20° C. freezer andallowed to thaw. Samples were cut into 2 cm×2 cm sections and washed insterile PBS×3 to remove any freeze protectants. The samples were thenplaced BM side up onto microscope slides and allowed to slowly air dryat 4° C. for 24 hours. Samples were not fixed or critical point dried.After drying the sample was ready for imaging.

H&E and Immmunoperoxidase staining of BM proteins—Collagen IV, CollagenVII and Laminin 5.

H&E staining to look at morphological features of ADM BM andimmunoperoxidase staining to confirm presence of the BM was carried outusing standard laboratory protocols. Immunoperoxidase staining wasperformed for collagen IV, collagen VII and laminin 5. It was importantto stain for BM proteins so that it was certain that all imaging andanalysis was actually conducted on the BM. It is clear in practice tosee increased staining along the top of ADM where there is thin strip oftissue. This is the basement membrane and it has stained positively forthree of the main basement membrane proteins; collagen 4, collagen 7 andlaminin 5. These images confirm the presence of the basement membrane,superior to ADM and confirm that all the measurements and analysisconducted are of the ADM BM.

Methods of Preparing Implant Surfaces of the Invention Example1—Production of ADM BM PDMS F—Fabricated Implant Surface by GrayscaleLithography of Acellular (Decellularised) Dermal Matrix Pattern andReproduction in Silicon by Modified Deep Reactive Ion Etching (DRIE) toCreate a Template Before Creating Stamps of ADM BM Pattern in PDMS

Imaging the ADM BM Surface

The basement membrane (BM) side of the ADM BM surface is distinguishedfrom dermal side through visually checking the tissue for roughness anda buff-colour. Further, BM characteristically repels water and thecontact angle of the water is higher than on BM side than PD side. Thesamples were then placed BM side up onto microscope slides and allowedto slowly air dry at 4° C. for 24 hours. Over one hundred 90 μm×90 μm²AFM scans were conducted in different areas of the ADM sample, on anumber of different samples.

Preparing an Electronic File from Height Information of Grayscale AFMImages

The raw images from the AFM database were loaded into the followingscanning probe analysis software, NanoScope Analysis. A plane fit (0-2orders) was applied to all images. The images were then exported asbitmaps (BMP's). The BMP's were loaded into the following open sourcescanning probe analysis software Gwyddion (http:/gwyddion.net/) toconvert to 8 bit grayscale BMP's. Each AFM image was re-scaled as 180pixels. The ADM montage was created using an open source imagingsoftware to stitch together various AFM images of ADM, which were chosenbased upon their superior image quality. AFM images of ADM BM werestitched together based upon similarities in height at the edges of theimages so that images could be blended without leaving stitch lines TheADM montage was converted to an 8 bit grayscale image, which consists of256 grayscale levels, which could be read by a laserwriter (MicrotechLaserwrited LW405, but other laserwriters can be used). FIG. 1 shows anexemplary 3D rendered grayscale image montage of the ACD.

Exposing ADM Pattern into Photoresist

Maskless grayscale photolithography was performed using a laserwriter.The 8-bit grayscale image prepared as above, was loaded into thelaserwriter software and the exposure dose for each pixel assigned.Vertical features of the ADM were scaled down for incorporation into theS1813 photoresist as it is thin (1.3 microns) whereas vertical featuresof the ADM topography were larger than 2 microns. A 2 cm×2 cm plainsilicon wafer was sonicated for 5 minutes in acetone, distilled waterand isopropyl alcohol (IPA). The sample was then dried with nitrogen gasand dehydrated on a hot plate set at 130° C. for 10 minutes. Immediatelyafter removing from the hotplate the sample is placed into the spinnerand hexamethlydisilazane (HMDS) is applied to the surface. It is thenleft to rest for 10 seconds and spun at 4000 rpm for 60 seconds. S1813photoresist was spin coated onto silicon wafers at 3000 rpm for 60seconds, after application of HMDS, followed by soft bake at 72° C. for2 minutes. The prepared grayscale BMP is loaded into the Laserwriter andthe pixel size is set at 0.5 um in X and Y, which is the smallestpossible pixel size. (A 180×180 pixel image will therefore be 90×90 umafter exposure). Using a gain of 5.5 and bias of 1, exposure to UV leadsto degradation of the resist as performed by the Laserwriter. Theexposure is developed in MF319 for 40 seconds with gentle agitationbefore 40 seconds in IPA. A final rinsing in distilled water wasperformed and the wafer was dried with nitrogen gas.

Modified Deep Reactive Ion Etching (DRIE)

Deep reactive ion etching using a modified Bosch process with scale upof the vertical features allowed permanent fixture/transfer of the ADMsurface topography in the silicon template.

Grayscale etching recipe: Etch selectivity: 10:1. Step Pressure RF ICPSF₆ C₄f₈ 0₂ Step time (mTorr) power power (Sscm) (Sscm) (Sscm) Etch 3 105 300 100 5 0 0₂ Etch 3 10 5 300 0 0 30 Deposition 4 10 5 300 5 100 0Repeats 80-100

Manufacture of PDMS Medical Grade Casts

The PDMS used was MED-6215 (Nusil, Calif., U.S) optically clear PDMSelastomer. The PDMS came in two parts; a viscid PDMS elastomer (Part A)and a runny platinum curing agent, “Part B”, which were mixed togetherat a ratio of 10:1 by weight. The PDMS was de-gassed in a desiccator for1 hour to remove any bubbles. The silicon wafer was vapor treated with asilanizing agent (TMCS) for 10 mins in a desiccator under vacuum, inorder to ease the release of PDMS from the silicon wafer. PDMS was spunat 130 rpm on to silicon wafer containing the 1.5 cm×1.5 cm exposure ofADM montages. Spinning at 130 RPM produced PDMS with a thickness ofapproximately 450-600 um, which is similar to the thickness ofcommercially available silicone breast implant shells and could also bepeeled away easily from the silicon wafer without fragmenting. PDMS wasspun onto wafers using a programmable resist spinner. A curing step wasrequired to crosslink and harden the PDMS and therefore the PDMS wasbaked in an oven at 80° C. overnight. The 1.5×1.5 PDMS stamp was thencut out using a scalpel, which creates the finished PDMS stampcontaining the surface features of ADM reproduced in it.

AFM imaging was used to acquire the respective images for reproductionvia the fabrication method. However, higher or lower resolution imagedata may be obtained using alternative imaging technologies. Anysuitable contact or non-contact profilometers can be used, suitably toproduce grayscale images where each pixel correlates to a given height.Thus, surfaces having a variety of macro, micro and nano surfacefeatures according the invention may be produced. In addition, since thefabrication method allows for production of digital information of thesurface, the surface information can thus be processed (such as tofilter out certain features) to produce a variety of surfacetopographies, including those according to the invention.

Example 2—Creation of ADM BM C—Casting of Acellular Dermal Matrix inPDMS

Preparation of Acellular Dermal Matrix (ADM)

ADM washed in sterile deionised water ×3 and attached BM side up ontomicroscope slide and allowed to slowly air dry for 24 hours at 4° C.Basement membrane (BM) side of ADM is distinguished from dermal sidethrough visually checking the tissue for roughness and a buff-colour.Further, BM characteristically repels water and the contact angle of thewater is higher than on BM side than RD side.

Silanization

To allow easy peeling of PDMS from ADM BM, the ADM was silanized.Silanization performed by placing ADM in vacuum desiccator on asteel-meshed platform with 100 ul of the fluorosilane TMCS in a 3 cmdiameter petri dish beneath the ADM. The vacuum was pumped for 30seconds-1 min until the TMCS is visibly beginning to bubble andevaporate. At this stage the desiccator is held under vacuum for 10minutes to allow the TMCS to silanize the ADM surface. The ADM is thenready for casting.

Creation of Inverted Cast

PDMS is spun onto the ADM at 4000 RPM for 1 minute then left at roomtemperature (17° C.) for 48 hours. This step was repeated twice. ThePDMS was carefully peeled off ADM and place on a cured square of PDMS ona clean microscope slide, pattern facing upwards. The PDMS was thencured overnight at 120° C.

Creating Final Cast of ADM

PDMS is again silanized with TMCS using method outlined above. PDMS(Mentor Corporation) is spun onto the inverted ADM PDMS pattern at 4000RPM for 1 minute then baked at 25° C. for 48 hours. This step wasrepeated twice. PDMS was then spun onto the surface at 2000 RPM for 1minute followed by baking at 60° C. for 1 hour. This was repeated twice.PDMS was again spun at 1000 RPM for 1 minute then baked at 60° C. for 30minutes, followed by spinning of more PDMS at 500 RPM for 1 minute thencuring overnight at 120° C. This resulted in an implant thickness of450-600 um thick (Same range as commercially available implants. Theresulting ADM BM PDMS cast was then characterised as described below andsubject to biological cellular assays as described below.

Comparison of Fabrication Method to Casting Method

The main benefit of the casting method is that it allows for moreprecise replication of the topographical features of ADM BM. Inaddition, the casting method allows replication of the full range offeatures of the ADM BM topography from macro to nano scale (see FIGS.26A-26C for a comparison of natural ADM BM tissue surface and ADM BM Cas cast according to the method above showing a close reproduction ofthe natural features in the cast implant surface).

In contrast, the fabrication method as described above is limited by therelevant range on the AFM scanner and/or the lithography technique used.Thus, lower resolution images were produced compared to the castingmethod but the method is more versatile and adaptation of the digitalX,Y,Z information can provide a variety of surface topographies. FIG. 25shows that the features of natural ADM BM are recreated fairlyaccurately using the grayscale fabrication method, although not at thesame level of accuracy as the casting method. Use of Electron Beam(E-beam) Lithography to create the pattern should however allow thereproduction of features that are <50 nm in lateral resolution.

Characterisation of Implant Surfaces (Topography and Roughness)

Characterization and Quantification of Topographical Features BM/PD inAcellular Dermal Matrix (ADM) and Commercially Available Implants

Preparing ADM for Characterisation

The ADM was removed from storage in −20° C. freezer and allowed to thaw.Three different samples of ADM were used for analysis. Tissue wasremoved from foil packaging and taken out of gauze. Samples were cutinto 2 cm×2 cm sections and washed in sterile PBS×3 to remove any freezeprotectants. Basement membrane (BM) side of ADM is distinguished fromdermal side through visually checking the tissue for roughness and abuff-colour. Further, BM characteristically repels water and the contactangle of the water is higher than on BM side than PD side. The sampleswere then placed BM side up onto microscope slides and allowed to slowlyair dry at 4° C. for 24 hours. Samples were not fixed or critical pointdried. Samples were then ready for measurement with optical microscopy,atomic force microscopy (AFM), environmental scanning electronmicroscopy (ESEM), Optical 3D profiler, White Light Interferometry (WLI)and profilometry.

Preparation of Comparative Examples

Commercially available implants used for comparison were Mentor Smoothand Mentor Siltex (Mentor Corporation, Santa Barbara, Calif.). Squaresamples of 1 cm×1 cm were cut out from implants. 3 different sampleswere cut from each implant and 3 different implants were used, for atotal of 9 samples per implant type. Samples were sonicated in adetergent for 10 mins followed by DI water for 10 mins prior to beingair dried overnight. Mentor Siltex implants were too rough formeasurement with AFM and therefore Optical 3D profiling, White LightInterferometry and 3D laser scanning were used.

Characterisation of Surface Topographies

A) Characterisation of Surfaces of the Invention

ADM BM surfaces according to the invention were measured using BrukerIcon Dimension Atomic Force Microsope for scan sizes 90×90 um, 10×10 umand 1×1 um. Samples were imaged using ScanAsyst Air probes (nominalk=0.4 N/m). Imaging was conducted using ScanAsyst. PFT amplitude was150-100 nm, and PFT frequency was 1 kHz. Scan rate was 0.5 Hz. Imageswere analysed using NanoScope Analysis software. A plane fit (0-2orders) was applied prior to analysis.

AFM scans were performed at 3 different sizes. Ten images for each scansize were obtained at 90 um×90 um, 10×10 um and 1 um×1 um.

1 mm×1 mm scans of ADM BM were performed with a Bruker 3D OpticalProfiler and White Light Interferometer as the AFM could not performscans at that size.

ISO 25178 (Surface texture: areal) roughness analysis of each image wasperformed (using Bruker's NanoScope Analysis Software) and exported to aMicrosoft Excel spread sheet for calculation of mean roughness valuesand standard deviation. For calculating fractal dimension andautocorrelation lengths open source online scanning probe microscopyanalysis software Gwyddion was used. Matlab and Gwyddion were used forobtaining auto correlation functions of implant surfaces.

B) Comparative smooth implants (Mentor Smooth): all characterisation forthese implant surfaces were measured in the same way as for ADM BMsurfaces of the invention described above. Reference to smooth implantsin the biological methods below refers to comparative Mentor smoothimplant surfaces.

C) Comparative textured implants (Mentor Siltex): All measurements wereperformed with a Bruker 3D Optical Profiler, White Light Interferometerand 3D laser scanner, as the surface roughness of these implants isoutside the Z range of the AFM. Reference to textured comparativesurfaces in the biological methods below refers to comparative MentorSiltex implants.

ISO 25178 (Surface texture: areal) roughness analysis of each image wasperformed and exported to a Microsoft Excel spread sheet for calculationof mean roughness values and standard deviation. For calculating fractaldimension and autocorrelation lengths, open source online scanning probemicroscopy analysis software Gwyddion (discussed above) was used.

Data showing the ISO 25178 (Surface texture: areal) roughness values ascalculated above are depicted in FIGS. 24A-24B.

Comparison of Surface Characterisation Data

The surface microstructure of ADM BM and commercially available smoothand textured implants were comprehensively characterised using opticalmicroscopy, AFM, SEM, 3D profiling, WLI and profilometry. Roughnessmeasurements and topographical data were gathered using a variety ofexperimental techniques. All surfaces were measured on at least fourdifferent length scales to ensure complete capture of roughness andtopographical data and to allow comparison between them.

3D imaging and surface analysis reveal large differences in topographyand roughness between ADM BM and commercially available implants.Textured implants (Mentor Siltex, Sa=8.24 μm) were more than 20 timesrougher than ADM BM (Sa=0.48 μm) and around 400 times rougher thansmooth implants (Mentor Smooth, Sa=0.022 μm) at similar length scales

The Sa (arithmetic mean of values) of ADM BM varied at different imagesizes and ranged from 6.7 μm at 1 mm×1 mm scans down to 5.84 nm for a 1μm×1 μm scan. The Sa of smooth implants ranges from 82.65 nm at 1 mm×1mm to 4.36 nm at 1 μm×1 μm scans while textured implants range from41.73 μm at 1 cm×1 cm to 8.24 at 100 μm×100 uμm. ADM BM surfacetopography according to the invention is therefore considerably rougherthan smooth implants while significantly less rough than texturedimplants.

The Sz (Maximum height of the surface (Distance between maximum valleydepth and maximum peak height) of ADM BM ranges from 43 μm for 1 mm×1 mmscans to 46.9 nm for 1 μm×1 μm scans. This is in comparison to texturedimplants where the Sz ranged from 273 μm for 1 cm×1 cm scans to 40 μmfor 100 μm×100 μm scans. Further, smooth implants Sz ranged from 1.2 μmfor 1 mm×1 mm scans to 46.64 nm for 1×1 μm scans. The maximum featureheights on textured implants are therefore considerably larger than onADM BM surfaces of the invention and comparative smooth implants whileADM BM contains maximum feature heights that are larger than those onsmooth implants at every length scale.

Ssk (Skewness) describes the degree of symmetry of surface heights aboutthe mean, i.e. whether a surface possess a predominance of either peaksor valleys. A negative Ssk indicates a predominance of valleys and apositive Ssk indicating a predominance of peaks. Measured ADM BM surfaceSsk (skewness) was found to be approximately 0 at all length scalesranging from 0.14 at 1 mm×1 mm scans to −0.04 at 1 μm×1 μm scans. TheSsk values are all very close to 0 indicating neither a predominance ofpeaks nor valleys but an equal contribution of both. This is in contrastto the varying Ssk value for textured implants (Mentor Siltex) of 0.57for 1 cm×1 cm scans to −0.02 for 100 μm×100 μm scans indicatingvariation in peak to valley ratio at different length scales, but with apredominance of peaks at increased length scales. Comparative smoothimplants (Mentor Smooth) had a positive skewness at length scalesranging from 3.58 for 1 mm×1 mm scans to 0.11 to 1 μm×1 μm indicating apredominance of peaks at all length scales.

Sku (kurtosis) describes the likelihood of a surface having a featurewhich is significantly deviated from the mean. Excess Sku values will beused to describe Sku values throughout these results. It is calculatedby Sku-3. A surface which contains features that significantly andabruptly deviate from the mean will have a positive Sku (Sku>0) where asa surface which is gradually varying will have a negative Sku (Sku<0). Asurface which contains a bell-shaped curve of normal distribution has aSku of 0. A surface having a Sku of 3 shows a Gaussian distribution.Thus, a surface having an excess kurtosis (Sku-3) of zero shows Gaussiandistribution.

ADM BM according to the invention possesses excess kurtosis (Sku) valuesof close to 0 at all length scales indicating that surface features ofADM BM are normally distributing. ADM BM possess an exess Sku of −0.15for 1 mm×1 mm scans down to −0.1 for 1 μm×1 μm scans. This is incontrast to the positive Sku values of smooth implants at all lengths,ranging from 26.7 for 1 mm×1 mm scans to 0.93 for 1 μm×1 μm scans. Thisindicates that smooth implants (Mentor Smooth) are predominantly flatbut contain repeating and random small peaks on the surface. Thenegative Sku values of textured implants (Mentor Siltex), ranging from−1.88 for 1 cm×1 cm scans to −3.21 for 100 μm×100 um scans indicate amore gradually varying surface with predictable variations from themean.

Considering Ssk and Sku values together it may be said that the ADM BMsurface measured is a self-similar surface with similar Ssk and Skuvalues at all length scales. It has a roughly equal distribution ofpeaks and valleys that are gradually varying. The surfaces fit a normaldistribution and the graph would be shaped like the “bell curve”.Textured implants can be described as a being macroscopically rough withrepetitive and repeatable features with a slight predominance of peakswhich are gradually varying and predictable. Smooth implants aremacroscopically smooth surfaces which contain features which arepredominantly peaks which abruptly deviate from the mean and are random.

The fractal dimension (FD) of ADM BM at all size scans is approximately2.3, ranging from 2.37 for 1 mm×1 mm scans to 2.29 for 1 um×1 um scans.A fractal dimension of a plane is 2 and a cube is 3 therefore thefractal dimension of ADM BM indicates a planar surface with 3D featureson it. As the FD is consistent across all scan sizes it suggests thatthe same surface topography is present but at different scales; macro,micro and nano-scale topographies. This is classical self-similaritycommonly found in nature.

The FD of textured implants varies with scan size. It ranges from 2.81for 1 cm×1 cm scans to 2.05 for 100 um×100 um scans. This indicates thatit goes from being a 3D surface to one that can nearly be consideredplanar. This is because the topography and roughness of texturedimplants vary greatly depending on the area over which they aremeasured; containing distant macroscopic features but is mostly smoothat the micron and nano-scale level.

The FD of smooth implants also varies with scan size. It ranges from2.07 for 1 mm×1 mm scans to 2.36 at 1×1 um scans (and 2.59 at 10×10 umscans). This indicates that smooth implants are practically planar andflat when measured over a large area and becomes gradually rougher onthe micron and nanoscale at smaller scan sizes. It can be described asmacroscopically and microscopically smooth at large scan sizes andnanoscopically rough at very small scan sizes.

Production of Surfaces Having Variations of the Values Measured

Modification of autocorrelation functions derived from thesemeasurements could be used to filter the Gaussian distribution offeatures to create a model surface of ADM BM with a fractional dimensionof 2.3.

General Biological Methods

Isolation of Breast Derived Fibroblasts (BDF's) from Breast Tissue andCell Culture

Breast derived fibroblasts (BDF's) were used for all studies torepresent the cells which will encounter the implants if they wereinserted into breasts in vivo. Informed consent was taken from eachpatient undergoing surgery and written ethical approval was gained fromlocal Ethical Committee. It has been shown that the site of tissueharvest contains fibroblasts, which are site specific. Fibroblasts fromdifferent body sites have different genotypic and cytokine profiles soit was important to use breast derived fibroblasts to most accuratelyre-create the in vivo environment in vitro and also allow strongerconclusions to be made as to how the effect of the different implants oncells may be realised in vivo (clinical application).

Primary cell culture of breast gland and connective tissue, to obtainBDF's, was performed. Cells were grown in T75 tissue culture plastic(TCP) flasks (Corning Incorporated, USA) in growth media containingDulbeccos Modified Eagle Medium (DMEM) (Sigma Aldrich, Aldrich, UK)supplemented with 10% FBS (PAA, Austria) Penicillin (100 units/ml),streptomycin (100 units/ml) and L-Glutamine (2 mM, PAA Austria). Theyare incubated at 37° C. in humidified in 5% CO₂ air. Growth media ischanged every 48 hours and cells passaged at 70-90% confluence. AllBDf's used in the following experiments are of passage 3 in an effort toretain the cells innate genotypic and phenotypic characteristics beforethey're removed with excessive passaging.

Cell Attachment and Cell Proliferation (MTT Assays)

For cell attachment and proliferation rate studies, 10,000 cells perwell (24 well plate) were seeded. Each experiment was performed threetimes, in triplicates. MTT assays were performed on ADM BM F accordingto the invention (prepared by the grayscale fabrication method describedabove), ADM BM C according to the invention (prepared by the castingmethod described above), comparative smooth implants, comparativetextured implants, comparative tissue culture plastic (TCP) andcomparative collagen.

Cell attachment experiments were performed using an MTT assay (Cellproliferation Kit 1 (MTT), Roche, Mannheim, Germany), as permanufacturer instructions. It is a colorimetric assay in which thetetrazolium salt MTT gets cleaved intracellularly within viable cells,which after the cells have been solubilized, produces a purple formazandye. This can be measured using a microplate reader at a wavelength ofbetween 570-650 nm and after background has been removed an absorbance(optical density) value can be obtained.

Cell Survival (LDH Assay)

Cell survival was determined using the lactose dehydrogenase (LDH)enzyme, which is released by damaged cells into the growth media. Thelevels of LDH released by damaged cells into the growth medium can bemeasured as per manufacture instructions, using a micro-plate reader andmeasured between a wavelength of 490-660 nm (Cytotoxicity Detection Kit,Roche Mannheim, Germany).

RNA Extraction, cDNA Synthesis and Quantitative Real Time PolymeraseChain Reaction (QRT-PCR)

Following BDF culture on different surfaces, cells were collected inTRIzol buffer (Invitrogen, UK). RNA extraction, cDNA synthesis andQRT-PCR were carried out to manufacturers instructions, using standardprotocol in our laboratory and as described previously [Shih 2012]&[Shih 2010]. RNA concentration and purity were analysed on NanoDrop2000c (Thermo Scientfic, Rockford, Ill.). RNA concentration wasnormalized prior to cDNA synthesis. cDNA synthesis was carried out usingqScripts cDNA synthesis kit (Quanta Biosceinces, Gaithersburg, Md.).QRT-PCR was performed on LightCycler 480 machine (Roche Diagnostics,Germany), as described previously [Shih 2010]& [Syed 2011]. Primers andprobes used for QRT-PCR are shown in table below. Delta CT values werecalculated by subtracting averaged RPL32 (reference gene) CT values fromaveraged CT values of target gene. Relative gene expression levels werecalculated by using 2⁻ΔΔ^(CT) method.

TABLE 1 genes and primers used for QRT-PCR Universal Prober Target genePrimer Sequence Number Proliferating cell  Left: tggagaacttggaaatggaaa#69 nuclear antigen (PCNA) Right: gaactggttcattcatctctatggVinculin (VCL) Left: ctgaaccaggccaaaggtt #89 Right: gatctgtctgatggcctgctInterleukin 8 (IL8) Left: agacagcagagcacacaagc #72Right: atggttccttccggtggt Tumour necrosis factorLeft: agcccatgttgtagcaaacc #79 alpha (TNF-α) Right: tctcagctccacgccatt

Immunocytochemistry

Immunocytochemistry was performed on BDF's for vinculin, F-Actin andDAPI. BDF's cultured on different surfaces were fixed in 10% Neutralbuffered formalin (NBF) for 1 hour, washed in PBS and permeabilised in0.5% Triton-X 100 for 25 minutes. Cells were washed again with PBS andblocked in blocking solution (1% BSA) for 1 hour at room temperature ona shaker at 55 RPM. After washing, cells were incubated overnight at 4°C. with Mouse-Monoclonal Anti-vinculin primary antibody (SPM227,ab18058, Abcam, UK), at a dilution of 1:50 in PBS. The following stepsare performed in the dark. Cells are washed with PBST (0.1% tween inPBS) and then incubated with the secondary antibody Anti-rabbit AlexaFluor-488 dye (Invitrogen, UK) in a 1:200 dilution on a shaker at 55 RPMfor 1 hour at room temperature, wrapped in foil. After washing in PBST,cells were incubated with Rhodamine phalloidin stain (1:200)(Sigma-Aldrich, UK) for 45 mins at room temperature. Cells are againwashed with PBST before incubate with DAPI (1:500) (Invitrogen, UK) for15 minutes at room temperature. Surfaces were washed with PBST, mountedwith Prolong gold (Invitrogen, UK) and stored in cold room, wrapped infoil. Surfaces were visualised on an upright immunofluorescencemicroscope and images recorded. (BX51, Olympus UK Ltd)

SEM

BDF's that had been cultured on different surfaces were fixed in 10%Neutral buffered formalin (NBF) for 1 hour. They were then dehydrated ingraded, increasing alcohol concentrations of 50%, 60%, 70%, 80%, 95% and100%×2, for 10 mins each. Surfaces were dried, sputtered with gold andthen immediately imaged using FEI SEM+ESEM.

Statistical Analysis

All experiments were performed three times, in triplicates. Allstatistical tests were performed using Prism 6 software. Relativeabsorbance (OD) values of the colorimetric MTT/LDH assays were used forcell attachment, cell proliferation and cell survival comparisons.Two-way ANOVA followed by Turkey post-hoc multi-comparison analysis wasperformed on cell attachment, proliferation rate and cell survival data.To determine the difference in gene expression between BDF's ondifferent surfaces the relative threshold cycle (C_(T)) was used,obtained from PCR. Relative gene expression was calculated using the2⁻ΔΔ^(CT) method and used for comparison. One way ANOVA followed byTurkey post-hoc multi-comparison analysis was performed on QRT-PCR data.A p value of less than 0.05 was considered as statistically significantin all experiments.

Cellular Response Data

In vitro evaluations of Breast Derived Fibroblast (BDF) cell attachment,proliferation, survival, genotype and phenotype on ADM BM F (fabricatedaccording to example 1) and ADM BM C (cast according to example 2) PDMSimplants according to the invention were performed. These data werecompared against data for conventional smooth and textured implantsinter alia and showed improved properties in all tested areas asmentioned below.

1) Cell Attachment

As seen in FIG. 2, cell attachment of BDF's was significantly greaterafter 2 hours on both ADM C and ADM F surfaces according to theinvention as compared to smooth and textured implants, which persistedup to the 6 hour time point. During the first 1 hr of cell culture therewas no observed difference in cell attachment between implant surfaces.However, by 2 hrs, significantly more BDF's had attached to ADM surfacesof the invention in comparison to smooth and textured implants. Thiseffect was observed through 4 and 6 hours, with most significantdifferences observed after 6 hours. After 6 hours, cell attachment ofBDF's on both ADM BM F and ADM BM C surfaces of the invention wassignificantly greater than on smooth (ADM BM F p<0.0001; ADM BM Cp=0.042) and textured (ADM BM F p<0.0001; ADM BM C p<0.0001) implants.Further, there was no significant difference between cell attachment onsmooth or textured implants after 6 hours, and no significant differencebetween ADM BM C and ADM BM F.

2) Proliferation Rate (MTT Assay)—Proliferation of BDF's on ADM C andADM F was Significantly Greater than on Smooth and Textured Implantsafter 24 Hours which Progressively Increased and Continued Up to 1 Week

As seen in FIG. 3, after 24 hours significantly increased cellproliferation was observed on ADM surfaces according to the invention incomparison to smooth (ADM BM F p=0.034; ADM BM C p=0.045) and texturedimplants (ADM BM F p=0.0034; ADM BM C p=0.0049), which althoughplateauing, was still significant at 48 hours. By 96 hours there was aclear increase in proliferation of BDF's on ADM surfaces in comparisonto smooth and textured which became most significant after 1 week (after1 week, ADM BM F vs. smooth p0.015; vs. textured <0.0001; ADM BM C vs.smooth p<0.0001; vs. textured p<0.0001). After 1 week, there wassignificantly increased BDF proliferation on smooth implants incomparison to textured (p0.0363). Further, after 1 week there wassignificantly increased proliferation of BDF's on the ADM BM C surfacein comparison to the ADM BM F surface (p0.0017). These data indicate bycomparison of the ADM BM F and ADM BM C the improved effect on cellularproliferation achieved by the novel sub-micro and nano-scale roughnessfeatures present in the ADM BM C surfaces (included in the ADM BM Csurfaces as a result of the increased resolution of the casting methodover the fabrication method of the invention described herein).

3) Cell Survival (LDH Assay)—BDF's on ADM Surfaces Showed Less Apoptosis(Increased Cell Survival) at Every Time Point

As seen in FIG. 4, LDH assay revealed improved BDF survival on ADMsurfaces according to the present invention at every time point incomparison to conventional smooth and textured implants. This was mostsignificant after 96 hours and continued up to 1 week (at 1 week, ADM BMF vs. smooth <0.0001; vs. textured <0.0001; ADM BM C vs. smoothp<0.0001; vs. textured p<0.0001). In addition, increased cell death wasobserved in BDF's cultured on textured implants in comparison to smoothimplants after 1 week (p0.0023). Further, at 1 week there wassignificantly increased cell survival of BDF's on ADM BM C surface incomparison to ADM BM F surface (p0.043). These data again indicate bycomparison of the ADM BM C and ADM BM F data the improved effect oncellular proliferation of the sub-micro and nano-scale surface roughnessfeatures present in the ADM BM C surfaces as a result of the increasedresolution of the casting method over the fabrication method of theinvention described herein.

4) QRT-PCR—Changes in Gene Expression of BDF's Cultured on DifferentImplant Surfaces for 96 Hours

a) PCNA

PCNA (proliferating cell nuclear antigen) is a gene, which becomeshighly expressed during DNA synthesis and DNA repair. Cells which areproliferating (Undergoing mitosis) are constantly synthesizing new DNAprior to replication. Therefore, PCNA is a measure of cell proliferationlevels.

As seen in FIG. 5, PCNA was significantly up-regulated on ADM surfacesaccording to the present invention in comparison to smooth and texturedimplants (ADM BM F vs. smooth 0.034; vs. textured 0.0014; ADM BM C vs.smooth p<0.0001; vs. textured p<0.0001). Further, PCNA was significantlyup-regulated in BDF's cultured on ADM BM C in comparison to ADM BM F(p0.0069). In addition, there was significant difference in PCNAexpression of BDF's on smooth and textured implants.

This PCNA data correlates well with the cell proliferation data gatheredfrom MTT experiments, e.g. reinforcing the improved proliferation datafor ADM BM C material observed over ADM BM F.

b) Vinculin

Vinculin is a membrane-cytoskeletal protein that plays an essential partin the focal adhesions formed between cells and there environment. Firmfocal adhesion formation is important for cell attachment and subsequentcell spreading, migration and proliferation. Further,mechano-transduction (the method by which cells are able to convertmechanical stimulus from their environment e.g. implant surface or ECMand turn into chemical activity, such as a change in secretion of acytokine) is crucial to cell function and response to environment.

As seen in FIG. 6, vinculin was significantly up-regulated on ADMsurfaces according to the invention in comparison to smooth and texturedimplants (ADM BM F vs. smooth 0.0092; vs. textured 0.0008; ADM BM C vs.smooth p<0.03; vs. textured p<0.01). Further there was no significantdifference between BDF's cultured on ADM BM F in comparison to ADM BM C(p0.19).

Up-regulation of the vinculin gene in BDF's cultured on ADM BM surfacescorrelates with cell attachment experiments above.

c) IL8

IL8 (Interleukin 8) is an acute inflammatory chemokine and plays a keyrole in recruiting neutrophils to the wound after injury. It isassociated with many inflammatory conditions such as rheumatoidarthritis and psoriasis. It also plays a role in angiogenesis, and isassociated with a number of fibrotic conditions such as cystic andpulmonary fibrosis. It has previously been found to be up-regulated incontracted breast capsules [Kyle 2013].

As seen in FIG. 7, IL8 was significantly down-regulated on ADM implantsurfaces of the invention in comparison to smooth and textured implants(ADM BM F vs. smooth p<0.0001; vs. textured p<0.0037; ADM BM C vs.smooth p<0.0001; vs. textured p<0.0001). There was no significantdifference IL8 expression between BDF's cultured on ADM BM F incomparison to ADM BM C (p0.45). Lastly, there was a significantdown-regulation of IL8 in BDF's cultured on textured implants incomparison to BDF's on smooth implants (p0.0043).

d) TNF Alpha

TNF-alpha (Tumour necrosis factor alpha) has been shown to be apro-inflammatory cytokine that stimulates the acute phase reaction. TNFalpha has been shown to be associated with capsular contractureformation in a number of studies, see e.g. [Tan 2010]& [D'Andrea 2007].

As seen in FIG. 8, TNF alpha was significantly down-regulated on implantsurfaces of the invention in comparison to smooth and textured implants(ADM BM F vs. smooth p<0.0001; vs. textured p<0.0001; ADM BM C vs.smooth p<0.0001; vs. textured p<0.0001). Further, TNF alpha wassignificantly down regulated in BDF's cultured on ADM BM C in comparisonto ADM BM F (p0.023). Lastly, there was a significant down-regulation ofIL8 in BDF's cultured on smooth implants in comparison to BDF's ontextured implants (p<0.0001).

5) Immunofluorescence—Cell Morphology and Phenotype

Example: BDF's on ADM BM C (in the initial colour images, Red=F-Actin,Green=vinculin and Blue=Dapi. White circles highlight vinculin stainingand focal adhesion formation

As seen in FIGS. 9A-9B, immunofluorescence images revealed specificfocal staining of vinculin in BDF's on ADM BM surfaces. The focaladhesion has the characteristic shape of the focal adhesions offibroblasts, with localised and defined staining of vinculin (asidentified by white circles). The raw colour data show many green“streaks” which are each a focal adhesion point where the BDF hasattached to a feature on the ADM BM surface. These images reveal thatBDF's on ADM BM surfaces have attached well, and have subsequentlyspread to develop typical fibroblast morphology.

Comparative Example: BDF's on Mentor Smooth Implants (in the InitialColoured Images: Red=F-Actin, Green=Vinculin and Blue=Dapi)

As seen in FIGS. 10A-10B, immunofluorescence images of BDF's on smoothimplants revealed diffuse and non-specific staining of vinculin in focaladhesions. The focal adhesions are poorly formed and can't be clearlydemarcated. The cells have aggregated on the surface of the smoothimplant and have preferentially bound to each other (through cadherins)instead of forming focal adhesions with the implant beneath. The cellsand aggregates have a round morphology, typical of poor cell attachment.Aggregated cells can exhibit a stressed cell phenotype. These imagesreveal that BDF's on smooth implant surfaces have attached poorly, andsubsequently are unable to spread and are therefore unable to developtypical fibroblast morphology.

Comparative Example: BDF's on Mentor Siltex Textured Implants (in theInitial Coloured Images: Red=F-Actin, Green=Vinculin and Blue=Dapi)

As seen in FIG. 11, immunofluorescence images of BDF's on texturedimplants again revealed mostly diffuse and non-specific staining ofvinculin in focal adhesions. However, some focal adhesions can beclearly demarcated indicating some stable focal adhesion formation(identified by white rectangles in FIG. 11). The cells have been able tospread to an extent, however, as shown in the FIG. 11 image, it appearsthat the cells are wedged within the valleys between the steep noduleson the textured implant surface. They are therefore unable to spread,migrate or proliferative effectively and are restricted by the steepsidewalls. It is reasonable to expect that the up to 9 cells observed tobe wedged within the bottom of the valleys will experience “contactinhibition”. This may lead to a stressed cell phenotype. These imagesreveal that BDF's on textured implant surfaces have become wedged withinthe bottom of valleys between implant nodules, and although show somesigns of focal adhesion formation and cell spreading they are restrictedand inhibited from spreading and proliferating optimally.

Comparative Example: BDF's on Tissue Culture Plastic (in the InitialColoured Images: Red=F-Actin, Green=Vinculin and Blue=Dapi). WhiteCircles Indicate Vinculin Staining within Focal Adhesions

As shown in FIG. 12, BDF's cultured on TCP show typical fibroblastphenotype in vitro. Strong staining for vinculin within local adhesionsis present, which is abundant and clearly defined (identified by whitecircles). This indicates strong focal adhesion formation and allows thecell to spread effectively. Significant cell attachment andproliferation can be observed by number of cells present.

Comparative Example: BDF's on Collagen (in the Initial Coloured Images:Red=F-Actin, Green=Vinculin and Blue=Dapi)

As shown in FIGS. 13A-13B, BDF's cultured on collagen show typicalfibroblast phenotype in vitro. Strong staining for vinculin within localadhesions is present, which is abundant and clearly defined (indicatedby white circles). Staining for vinculin is even more prominent in cellson collagen than cells on TCP. This indicates strong focal adhesionformation and allows the cell to spread effectively. Cells have spreadmore widely on collagen than cells on TCP. The presence of collagenappea's to have promoted significant cell attachment and cell spreading,even when compared to cells on TCP.

6) SEM—Cell Morphology

Example: BDF's on ADM BM C after 6 Hours.

As Seen in FIG. 14, after 6 hours BDF's on ADM BM C according to theinvention have attached and are beginning to spread on the surface(white circle indicates a BDF which is displaying typical fibroblastspread morphology)

FIG. 15 shows Scanning Electron Microscope (SEM) images of BDF's on anADM BM surfaces according to the invention after 24 hrs (A) and 48 hours(B,C and D). It is clear to see the spread morphology of the cells onthe ADM surface (white circles indicate BDF's which are displayingtypical fibroblast spread morphology).

DISCUSSION

ADM BM surface has been comprehensively characterized using a variety ofimaging and measuring instruments. Commercially available smooth (MentorSmooth) and textured (Mentor Textured) implants have also beencharacterized using the same methods, and have been compared to ADM BM.Extensive quantitative and qualitative data on all surfaces has beengathered, and a number of significant and striking differences betweensurfaces has been elicited.

Two novel silicone surfaces inspired by ADM BM topography werefabricated. These surfaces were biologically evaluated in vitro,comparing the effect of ADM BM biomimetic silicone surfaces of theinvention in comparison to smooth and textured implants on BDF cellattachment, proliferation, survival and expression of a number genesassociated with cell attachment and proliferation (Vinculin and PCNA),in addition to the acute inflammatory response (IL8 and TNF alpha).

Based on the above data, ADM BM surfaces according to the inventionpromoted increased BDF attachment after 6 hours in comparison to smoothand textured implants; increased BDF proliferation from 24 hours up to aweek in comparison to smooth and textured implants; PCNA (A geneup-regulated in proliferating cells) was up-regulated in BDF's culturedon ADM BM surfaces according to the invention in comparison to smoothand textured implants, correlating well with the cell proliferation dataabove; QRT-PCR revealed gene expression of Vinculin (A protein whichforms an integral part of the focal adhesion complex and provides ameasurement of the number of cells attached to a surface and how wellthey have attached) was up-regulated in BDF's cultured on ADM BMsurfaces according to the invention in comparison to smooth and texturedimplants; gene expression of IL8 (A chemokine which plays a key role inthe acute inflammatory response) was down-regulated in cells cultured onADM BM surfaces according to the invention in comparison to smooth andtextured implants; gene expression of TNF-alpha (A chemokine which playsa key role in the acute inflammatory response) was down-regulated incells cultured on ADM BM in comparison to smooth and textured implants;immunofluorescence imaging of BDF's for vinculin, alpha smooth muscleactin and F-actin revealed that cells cultured on ADM BM surfacesaccording to the invention contained better formed and abundant focaladhesions than cells cultured on the comparative smooth and texturedimplants; BDF's on ADM BM surfaces according to the invention displayedmore phenotypic fibroblast morphology in comparison to BDF's on smoothand textured implants; SEM of BDF's on the different surfaces revealedthat BDF's on ADM BM surfaces according to the invention were morespread and had a typical morphological appearance of fibroblasts. Incomparison, BDF's on smooth implants were rounded.

These promising data indicate that biomimetic inspired breast implantsurfaces, based on ADM BM topography, may have a potential clinicalapplication of reducing the formation of capsular contracture andimproving cellular response in general, when compared to smooth andtextured implants, through improved cell-surface mediated foreign bodyreaction.

Specific nano-scale and micro-scale features have been shown to improvecell attachment, proliferation and migration. In addition, furtherdownstream responses such as gene expression and cytokine release havebeen shown to be altered by nano and micro scale topographies. A surfacesuch as ADM BM incorporates all of these features and is extremelyeffective at performing its roles, one of which is to promote cellattachment and migration of cells, particularly during wound healing andtissue regeneration. Thus, the biomimetic surface implants of theinvention show great promise in stimulating a favourable inflammatoryand tissue synthesis response in vivo.

ADM BM surface features include a range of sizes with micro andnanoscale features superimposed on top of the macroscale features. Inmorphological terms, it indicates the nano-topography of the BM on topof the more undulated and rough PD. This complete range of surfacefeature sizes is expected to confer beneficial properties to an implantthrough promoting initial cell adhesion and function whilst alsoencouraging tissue integration into the finely textured surface andprevention of capsular contracture.

The ADM BM surfaces produced by the casting method of the presentinvention while also containing features on the nano and sub-micronscale, which may influence cell adhesion and therefore all downstreamfunctions, also contains larger features which are 10's of microns largewhich may begin to influence tissue integration. Thus, ADM BM surfacesaccording to the invention are likely to show in vivo influence onfibrous capsule formation at a cellular and tissue level. This cannot besaid for either smooth or textured implants. Textured implants may beable to encourage tissue integration, implant stability and disruptionof parallel collagen bundle formation but they are not able to influencecell response. Further, smooth implants appear to be unable to performat either of these levels.

The present invention thus provides an extremely valuable contributionto the art in providing new biomimetic surfaces for incorporationgenerally in implants, particularly breast implants, which enablesignificantly improved profiles of cell attachment, proliferation,survival and expression of genes associated with cell attachment andproliferation compared to conventional prior art implant surface types,thus making the present surfaces excellent candidates for use inpreventing adverse cellular responses to implants when placed in thebody.

A number of patents and publications are cited herein in order to morefully describe and disclose the invention and the state of the art towhich the invention pertains. Each of these references is incorporatedherein by reference in its entirety into the present disclosure, to thesame extent as if each individual reference was specifically andindividually indicated to be incorporated by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a pharmaceutical carrier” includes mixtures of two or moresuch carriers, and the like.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by the use of the antecedent “about,” itwill be understood that the particular value forms another embodiment.

This disclosure includes information that may be useful in understandingthe present invention. It is not an admission that any of theinformation provided herein is prior art or relevant to the presentlyclaimed invention, or that any publication specifically or implicitlyreferenced is prior art.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and the spirit of the invention.

REFERENCES

-   [Schulte, V. A. 2009] Schulte, V. A. et al., Biomacromolecules 2009;    10(10):2795-2801.-   [van Kooten, T. G. 1998] van Kooten, T. G. et al. J Biomed Mater Res    1998; 43(1):1-14.-   [Rompen, E. 2006] Rompen, E. et al. Clin Oral Implants Res 2006;    17(52):55-67.-   [Barnsley, G. P. 2006] Barnsley, G. P. et al. Plast Reconstr Surg    2006; 117(7):2182-2190.-   [Harvey, A. G. 2013] Harvey, A. G., Hill, E. W., Bayat, A. Expert    Rev Med Devices 2013; 10(2):257-267.-   [Mendonca, G. 2008] Mendonca, G. et al. Biomaterials 2008;    29(28):3822-3835.-   [Barr, S. 2011] Barr S, Bayat A. Aesthet Surg J2011; 31(1): 56-67.-   [Barr, S. 2009] Barr, S., Hill, E. W., Bayat, A. Open access Journal    of Plastic surgery, Current Implant Surface Technology: An    Examination of Their Nanostructure and Their Influence on Fibroblast    Alignment and Biocompatibility, Volume 9, 16 Jun. 2009.-   [Davila, A. A. 2012] Davila, A. A. et al. Arch Plast Surg 2013;    40:19-27.-   [Salzberg, C. A. 2012] Salzberg, C. A. et al. Journal of Plastic,    Reconstructive & Aesthetic Surgery (2013) 66, 323-328.-   [Liu, D. Z. 2013] Liu, D. Z. et al. Annals of Plastic Surgery, 2013,    Volume 00, Number 00, pages 1-5.-   [Shih, B. 2012] Shih B, Bayat A. Comparative genomic hybridisation    analysis of keloid tissue in Caucasians suggests possible    involvement of HLA-DRBS in disease pathogenesis. Arch Dermatol Res.    2012; 304(3):241-9.-   [Shih, B. 2010] Shih B, McGrouther D A, Bayat A. Identification of    novel keloid biomarkers through profiling of tissue biopsies versus    cell cultures in keloid margin specimens compared to adjacent normal    skin. Eplasty. 2010; 10:e24.-   [Syed 2011] Syed F, Ahmadi E, Iqbal S A, Singh S, McGrouther D A,    Bayat A. Fibroblasts from the growing margin of keloid scars produce    higher levels of collagen I and III compared with intralesional and    extralesional sites: clinical implications for lesional    site-directed therapy, Br J Dermatol. 2011; 164(1):83-96.-   [Kyle 2013] Kyle D J T, Harvey A G, Shih B, Tan K T, Chaudhry I H,    Bayat A. Identification of molecular phenotypic descriptors of    breast capsular contracture formation using informatics analysis of    the whole genome transcriptome. Wound Repair Regen. 2013;    21(5):762-9.-   [Tan 2010] Tan K T, Wjeratne D, Shih B, Baildam A D, Bayat A. Tumour    Necrosis Factor-α Expression Is Associated with Increased Severity    of Periprosthetic Breast Capsular Contracture. Eur Surg Res. 2010;    45(3-4):327-332.-   [D'Andrea 2007] D'Andrea F, Nicoletti G F, Grella E, Grella R,    Siniscalco D, Fuccio C, et al. Modification of Cysteinyl Leukotriene    Receptor Expression in Capsular Contracture: Preliminary Results.    Ann Plast Surg. 2007; 58(2):212-214. ISO 25178-2: 2012(E).

1-27. (canceled)
 28. A breast implant comprising a textured surfacehaving: a mean surface roughness Sa value of from 2 μm to 12 μm at anarea scale of 1 mm×1 mm; a mean surface skewness Ssk value of from −0.7to +0.7 at an area scale of 1 mm×1 mm; a mean excess kurtosis value (Skuminus 3) of −1.0 to +1.0 at an area scale of 1 mm×1 mm; and a maximumpeak height to trough depth Sz value from 10 μm to 80 μm at an areascale of 1 mm×1 mm; wherein the textured surface comprises silicone. 29.The breast implant of claim 28, wherein the textured surface has a meansurface roughness Sa value of from 3 μm to 9 μm at an area scale of 1mm×1 mm.
 30. The breast implant of claim 28, wherein the texturedsurface has a ratio of average peak height to average trough depth offrom 2:3 to 3:2 at an area scale of 1 mm×1 mm.
 31. The breast implant ofclaim 28, wherein the textured surface has a mean excess kurtosis value(Sku minus 3) of from −0.7 to +0.7 at an area scale of 1 mm×1 mm. 32.The breast implant of claim 28, wherein the textured surface has a meanexcess kurtosis value (Sku minus 3) of from −0.5 to +0.5 at an areascale of 1 mm×1 mm.
 33. The breast implant of claim 28, wherein thetextured surface comprises polydimethylsiloxane.
 34. The breast implantof claim 28, wherein the textured surface has a root mean square heightSq value of from 4 μm to 15 um at an area scale of 1 mm×1 mm.
 35. Thebreast implant of claim 28, wherein the mean surface roughness Sa value,the mean surface skewness Ssk value, the mean excess kurtosis value (Skuminus 3), and the maximum peak height to trough depth Sz value at anarea scale of 1 mm×1 mm characterize a primary surface topography of thetextured surface, and wherein the textured surface has a secondarysurface topography superimposed on the primary surface topography, thesecondary surface topography having a mean surface skewness Ssk value offrom −1.0 to +1.0 at an area scale of 90 μm×90 μm.
 36. The breastimplant of claim 35, wherein the secondary surface topography has a meanexcess kurtosis value (Sku minus 3) of −1.5 to +1.5 at an area scale of90 μm×90 μm.
 37. The breast implant of claim 35, wherein the texturedsurface has a tertiary surface topography superimposed on the primarysurface topography and the secondary surface topography, the tertiarysurface topography having a mean excess kurtosis value (Sku minus 3) of−1.5 to +1.5 at an area scale of 10 μm×10 μm.
 38. A breast implantcomprising a textured surface having a primary surface topography and asecondary surface topography superimposed on the primary surfacetopography; wherein the primary surface topography has, at an area scaleof 1 mm×1 mm: a mean surface roughness Sa value of from 2 um to 12 um; amean surface skewness Ssk value of from −0.7 to +0.7; and a mean excesskurtosis value (Sku minus 3) of −0.5 to +0.5; wherein the secondarysurface topography has, at an area scale of 90 μm×90 μm: a mean surfaceskewness Ssk value of from −1.0 to +1.0; and wherein textured surfacecomprises silicone.
 39. The breast implant of claim 38, wherein theprimary surface topography has, at an area scale of 1 mm×1 mm, a maximumpeak height to trough depth Sz value from 10 μm to 80 μm.
 40. The breastimplant of claim 38, wherein the primary surface topography of thetextured surface has a mean surface roughness Sa value of from 3 to 9 μmat an area scale of 1 mm×1 mm.
 41. The breast implant of claim 38,wherein the secondary surface topography of the textured surface hasmean surface skewness Ssk value of from −0.7 to +0.7 at an area scale of90 μm×90 μm.
 42. The breast implant of claim 38, wherein the texturedsurface has a tertiary surface topography superimposed on the primarysurface topography and the secondary surface topography, the tertiarysurface topography having a mean excess kurtosis value (Sku minus 3) of−1.5 to +1.5 at an area scale of 10 μm×10 μm.
 43. A breast implantcomprising a textured surface having: a mean surface roughness Sa valueof from 3 μm to 9 μm at an area scale of 1 mm×1 mm; a mean surfaceskewness Ssk value of from −0.7 to +0.7 at an area scale of 1 mm×1 mm; amean excess kurtosis value (Sku minus 3) of −0.7 to +0.7 at an areascale of 1 mm×1 mm; a root mean square height Sq value of from 4 μm to15 μm at an area scale of 1 mm×1 min; a maximum peak height to troughdepth Sz value from 10 μm to 80 μm at an area scale of 1 mm×1 mm; and amean surface skewness Ssk value of from −0.9 to +0.9 at an area scale of90 μm×90 μm; wherein textured surface comprises silicone.
 44. The breastimplant of claim 43, wherein the textured surface has a mean excesskurtosis value (Sku minus 3) of −1.5 to +1.5 at an area scale of 10μm×10 μm.
 45. The breast implant of claim 43, wherein the texturedsurface has a mean excess kurtosis value (Sku minus 3) of −1.5 to +1.5at an area scale of 90 μm×90 μm.
 46. The breast implant of claim 43,wherein the textured surface has a mean surface roughness Sa value offrom 0.1 μm to 5 μm at an area scale of 90 μm×90 μm.
 47. A method ofmanufacturing the breast implant of claim 43, the method comprisingpreparing the textured surface by molding a silicone material over atemplate having a negative of the textured surface.